Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method

Abstract

RNA digestions catalyzed by many ribonucleases generate RNA fragments containing a 2′,3′-cyclic phosphate (cP) at their 3′-termini. However, standard RNA-seq methods are unable to accurately capture cP-containing RNAs because the cP inhibits the adapter ligation reaction. We recently developed a method named “cP-RNA-seq” that is able to selectively amplify and sequence cP-containing RNAs. Here we describe the cP-RNA-seq protocol in which the 3′-termini of all RNAs, except those containing a cP, are cleaved through a periodate treatment after phosphatase treatment, hence subsequent adapter ligation and cDNA amplification steps are exclusively applied to cP-containing RNAs. cP-RNA-seq takes ~6 d, excluding the time required for sequencing and bioinformatics analyses, such downstream assays are not covered in detail in this protocol. Biochemical validation of the existence of cP in the identified RNAs takes ~3 d. Even though the cP-RNA-seq method was developed to identify angiogenin-generating 5′-tRNA halves as a proof of principle, the method should be applicable to global identification of cP-containing RNA repertoires in various transcriptomes.

INTRODUCTION

The advent of next-generation sequencing technologies has largely unveiled the cellular transcriptome, leading to great advances in our understanding of gene expression, biogenesis mechanisms, and the molecular functions of coding and non-coding RNAs. The current standard RNA-seq methods, particularly those targeting small non-coding RNAs, include an adapter ligation step in which two different oligonucleotide adapters of known sequences are ligated to the 5′- and 3′-ends of a targeted population of RNA molecules. Any RNAs that fail to ligate with both adapters are not amplified through subsequent RT-PCR reactions, hence they do not appear in the resulting sequencing data. Because adapter ligation reactions require that the targeted RNA substrates contain a 5′-terminal phosphate (P) and a 3′-terminal hydroxyl group (OH), standard RNA-seq methods limit their utility for sequencing RNAs with terminal modifications that inhibit adapter ligation.

A 2′,3′-cyclic phosphate (cP) at the 3′-end of RNA is one such modification with which RNAs are not ligated to a 3′-adapter. RNA fragments containing a 3′-terminal cP are produced from RNA digestions catalyzed by many endoribonucleases such as the following from various organisms: tRNA splicing endonuclease ¹, inositol-requiring enzyme-1 (Ire1) ², RNase T₂ ³, RNase L ⁴, angiogenin (ANG) ⁵, MazF ⁶, general control protein 4 (GCN4) ⁷, placental protein 11 (PP11) ⁸, nidoviral uridylate-specific endoribonuclease (NendoU) ⁹, and Colicin D ¹⁰. Ribozymes ^11,12 and Mpn1 exonuclease ¹³ also form a cP-containing 3′-end in their cleaved RNA fragments. Such cP formations are not just the result of specific ribonuclease digestions, but cP formations themselves can have a functional significance: cP formations regulate RNA-protein interactions, RNA stability and turnover, and cell proliferation ^7,14–16. In tRNA splicing, 3′-terminal cP of 5′-exons is an important intermediate which is processed by a cyclic phosphodiesterase for ligation with 3′-exons ¹⁷.

cP-containing RNAs have been reported to have a physiological significance in various biological processes. For example, U6 small nuclear RNA (snRNA) is an evolutionarily-conserved component essential for mRNA splicing; U6 snRNA contains a 3′-cP that is formed by Mpn1 and enhances the affinity of the RNA for its interacting proteins ^13,14. Also belonging to cP-containing RNAs are the 5′-halves of tRNAs produced by ANG-mediated tRNA cleavage upon various stress stimuli, known as tRNA-derived stress-induced RNAs (tiRNAs) ¹⁸. These 5′-tiRNAs promote stress granule formation ¹⁹, inhibit translation ^18,20,21, and trigger cellular stress responses and apoptosis in neurodevelopmental disorders ²². Our recent study demonstrated the occurrence of a distinct class of ANG-generating tRNA halves, sex hormone-dependent tRNA derived RNAs (SHOT-RNAs), whose expression is promoted by sex hormones and their receptors in breast and prostate cancers ⁵. 5′-SHOT-RNAs, corresponding to 5′-tRNA halves, are cP-containing RNAs and have a functional significance in cell proliferation ⁵.

Given that cP-containing RNAs are expressed as functional molecules, capturing the whole repertoire of cP-containing RNAs would reveal significant biological events that have not been identified using the current standard RNA-seq methods. While an increasing number of RNA sequencing data are accelerating large-scale comparative analyses of transcriptomes and greatly contributing to the identification of significant RNA species in biological phenomena and diseases ^23,24, cP-containing RNAs remain a hidden component in such analyses. In the course of investigating SHOT-RNAs, we recently developed a method named “cP-RNA-seq” that is able to selectively amplify and sequence cP-containing RNAs ⁵. Here we describe a detailed cP-RNA-seq protocol that can be adapted for global exploration and identification of cP-containing RNAs in any RNA samples derived from various organisms, tissues, and cells.

Different reactivity of 3′-terminal phosphate and cyclic phosphate of RNAs to enzymatic treatments

The different reactivity of 3′-P and 3′-cP of RNAs to enzymatic treatments (Fig. 1) is utilized as the basis of cP-RNA-seq. The T4 polynucleotide kinase (T4 PNK) has 3′-terminal phosphatase activity that removes both a P and cP from the 3′-end of RNAs to form a 3′-OH end ²⁵. On the other hand, a widely-used phosphatase such as calf intestine phosphatase (CIP) removes a 5′-P and 3′-P but not a 3′-cP. Although CIP treatment alone cannot remove a 3′-cP, a combination treatment of acid, such as HCl, followed by CIP treatment can remove a 3′-cP because the acid treatment hydrolyzes a 3′-cP and converts it to a 3′-P ²⁶.

Figure 1.

A schematic representation of the reactivity of a 3′-P and a 3′-cP with enzymatic treatments. T4 PNK removes both a 3′-P and 3′-cP to form a 3′-OH end. A phosphatase such as CIP removes a 3′-P (and 5′-P) but not a 3′-cP. An HCl treatment followed by CIP treatment (HCl + CIP) can remove a 3′-cP because the HCl treatment converts 3′-cP to a 3′-P.

Overview of the protocol

The entire procedure of cP-RNA-seq is illustrated in Fig. 2. Gel-purified RNAs with approximate targeted lengths are treated with a phosphatase (CIP), followed by treatment with a periodate (NaIO₄). These two treatments disrupt 3′-ends of the RNAs that originally contain 3′-P and 3′-OH ends. The RNAs containing a 3′-cP survive the treatments and therefore are exclusively ligated to adapters after their cP is removed by T4 PNK treatment. Subsequent RT-PCR amplification and next-generation sequencing of the amplified cDNAs thus selectively identify the sequences of 3′-cP-containing RNAs. After identification, the actual presence of 3′-cP in the representative identified RNAs should be biochemically validated. For the validation, total RNA is treated with T4 PNK, CIP, or acid (HCl) followed by CIP treatment, and then Northern blots are performed to examine the mobility of the targeted RNA bands. The cP-RNA-seq procedure takes ~6 d, excluding the time required for sequencing and bioinformatics analyses. In addition, the biochemical validation takes ~3 d to complete.

Figure 2.

A schematic representation of the cP-RNA-seq procedure for selective amplification and sequencing of cP-containing RNAs. Total RNA is extracted from cell lines or tissues (Steps 1–10), and then the RNAs with targeted lengths are gel-purified using denaturing PAGE (Steps 11–27). The purified RNAs are subjected to CIP treatment (Steps 28–36), periodate treatment (Steps 37–40), and T4 PNK treatment (Steps 41–43). By using an Illumina kit, the treated RNAs are ligated to adapters (Steps 44–53) and their cDNAs are amplified by RT-PCR (Steps 54–59). The amplified cDNAs are gel-purified using native PAGE (Steps 60–72), and then subjected to Illumina next-generation sequencing.

Application of the protocol

For identification of 5′-SHOT-RNAs, we applied the cP-RNA-seq method to total RNA isolated from BT-474 breast cancer cells ⁵. We successfully obtained ~33 million reads aligned to the human genome, of which ~85% were actually derived from 5′-tRNA halves, indicating high specificity, sensitivity, and credibility of the method ⁵. We expect that the concept of the cP-RNA-seq method will be widely applicable for identification of cP-containing RNA repertoire in any cells or tissues from any organism and even in valuable clinical samples from diseased patients, provided RNA samples are available. Identification of cP-containing RNA repertoire would complement the information obtained using standard RNA-seq and may identify novel RNA processing events.

Comparison with other methods

To date, there is only one alternative sequencing-based method reported for identification of cP-containing RNAs. By utilizing Arabidopsis thaliana tRNA ligase whose ligation activity is specific to cP-containing RNAs, Schutz et al. specifically ligated an adapter to cP-containing RNAs and identified U6 snRNAs and specific tRNA fragments as cP-containing RNA species in human brain total RNA ²⁷. The method requires the recombinant tRNA ligase and the 2′-phosphotransferase, which they expressed and purified by themselves, involves many steps including 3-times gel-purifications, and yielded sequences whose genome mapping ratio was not high. The cP-RNA-seq method is efficient, specific, and only requires commercially available reagents as described below.

Limitations

The selective amplification of 3′-cP-containing RNAs is dependent on the periodate-mediated cleavage of any other RNAs with a 3′-OH end after CIP treatment. Because the requirement for periodate cleavage is the presence of a 2′,3′-diol structure of ribose, post-transcriptional ribose modifications that displace the diol structure will prevent the cleavage. To date, plant microRNAs (miRNAs) ²⁸, plant and animal shot-interfering RNAs (siRNAs) ^29,30, and animal PIWI-interacting RNAs (piRNAs) ^31–34 are known to possess a 2′-O-methyl ribose modification that displaces the diol structure. Despite the absence of 3′-cP, these modified RNAs would not be cleaved via a periodate treatment and therefore would be sequenced by cP-RNA-seq. This point should always be remembered, especially when 20–30 nt small RNAs are subjected to the cP-RNA-seq method.

Experimental design

RNA sample extraction and preparation (Steps 1–27)

The necessary quantity and quality of starting RNA materials will widely vary depending on the expression levels of the targeted cP-containing RNAs in the total RNA. For identification of 5′-SHOT-RNAs in BT-474 cells, we used 50–100 µg of total RNA as a starting material and gel-purified 30–50-nt RNAs from the total RNA using denaturing PAGE ⁵. Because we failed to amplify 5′-SHOT-RNAs directly from the total RNA without the gel-purification step (data not shown), it is recommended to concentrate the targeted cP-containing RNA species (if known) using gel-purification or column chromatography before enzymatic and chemical treatments of the RNAs. If the targeted RNA species are unknown and exploration of the cP-containing RNAs is the objective, depletion of at least ribosomal RNA using a kit such as Ribo-Zero rRNA Removal Kit (Illumina) and RiboMinus™ Eukaryote Kit for RNA-Seq (Life Technologies) would help to improve the amplification efficiency of cP-RNA-seq. Extraction of smaller RNAs using a kit such as mirVana (Life Technologies) would also be an option.

Enzymatic and chemical treatments of RNAs (Steps 28–43)

Gel-purified RNA populations are supposed to be a mixture of RNAs with various terminal phosphate states; i.e., either 5′-P/3′-OH, 5′-OH/3′-OH, 5′-P/3′-P, 5′-OH/3′-P, 5′-P/3′-cP, or 5′-OH/3′-cP. The CIP treatment is used to remove a P from both the 5′- and 3′-ends of the RNAs, although the state of cP is not changed. Eventually, the RNA pools contain two subgroups: one with 5′-OH/3′-OH and a second with 5′-OH/3′-cP. Subsequent periodate treatment cleaves the cis-diol group of the 3′-OH end of the former group to generate 2′,3′-dialdehydes that no longer serve as a substrate for adapter ligation ³⁵. The latter group survives periodate treatment because of the presence of a 3′-cP. Therefore, after removal of the 3′-cP and addition of a 5′-P by T4 PNK treatment, the latter group becomes the only RNA that bears a 5′-P and 3′-OH ends for 5′- and 3′-adapter ligations. These enzymatic and chemical treatments enable selective adapter ligation to the RNAs that originally contained a 3′-cP.

cDNA library preparation and next-generation sequencing (Steps 44–72)

The treated RNAs are subjected to the adapter ligation reaction, followed by the RT-PCR amplification of cDNAs for next-generation sequencing. When identifying 5′-SHOT-RNAs, as described in this protocol, we utilized an Illumina TruSeq Small RNA Preparation Kit ⁵. The Illumina kit can be substituted with any cDNA library preparation kit for next-generation sequencing as long as it is based on adapter ligations to RNAs. Sequencing and bioinformatics analyses of cP-RNA-seq data will depend on the goals of the investigator and on the target RNA species. Sequence analyses of 5′-SHOT-RNAs are described in Honda et al. ⁵.

Many RNA post-transcriptional modifications are known to affect reverse transcription ³⁶, which will cause biased sequencing results of heavily modified RNAs, such as tRNAs and their fragments. Recent studies have succeeded in removing key RT-impairing modifications, such as m¹A, using AlkB demethylase and thus in improving the efficiency and quality of the amplification and sequencing of heavily-modified RNAs ^37,38. Therefore, if the targeted RNAs are expected to contain modifications, pretreatment of the RNA samples with the demethylase would be an option for better efficiency and quality of cP-RNA-seq.

Controls

To ensure that the sequences obtained from cP-RNA-seq are derived from cP-containing RNAs and not from ribose-modified RNAs, our protocol calls for a negative control experiment that lacks two steps: CIP treatment and T4 PNK treatment. Because the “CIP/T4 PNK–” procedure can specifically amplify the modified small RNAs with the 5′-P/3′-ribose modification, the appearance of cDNA bands from the full procedure, but not from the negative control procedure as shown in Fig. 3, would strongly suggest that the amplified bands are derived from cP-containing RNAs. The cDNA band pattern also indicates complete periodate-mediated cleavage for the RNAs containing 3′-OH ends, which is the key to the cP-RNA-seq method.

Figure 3.

Selective amplification of mouse 5′-tRNA halves using cP-RNA-seq. Total RNA extracted from mouse spleen was subjected to cP-RNA-seq method. As shown by an arrow, the method amplified ~150–155-bp cDNA products corresponding to the targeted 5′-tRNA halves (5′-adapter, 55 bp; 3′-adapter, 63 bp; and therefore inserted sequences, ~32–37 bp). The cDNA bands were absent from the negative control procedure without CIP/T4 PNK treatment.

Biochemical validation of the presence of cP in the identified RNAs (Steps 73–96)

Because of the limitation discussed above, the actual presence of 3′-cP in the representative identified RNAs should be biochemically validated. For validation, total RNA is first treated with either T4 PNK, CIP, or acid (HCl) followed by CIP treatment. By subsequent Northern blot for targeted RNA, the difference in the band mobility due to the changed 3′-cP state is then analyzed. Because the HCl + CIP treatment removes 3′-cP but the CIP treatment alone cannot, as shown in Fig. 4, the upshift of the “HCl + CIP” band compared with the “CIP” band indicates the original presence of 3′-cP in the targeted RNAs. In addition, because T4 PNK removes 3′-cP, the “T4 PNK” band should be shifted up compared with the non-treatment band. This validation method is effective when the targeted RNAs are short enough for discriminating band mobility between the RNAs with and without cP/P. The presence of cP in long RNAs should be validated by a different method such as that using Poly-A-polymerase ^39,40.

Figure 4.

Biochemical validation of the presence of a cP in a mouse 5′-tRNA half. Mouse spleen total RNA was treated with T4 PNK, CIP, or acid followed by CIP treatment (HCl + CIP). NT designates non-treated sample used as a negative control. The treated or non-treated RNAs were subjected to Northern blots targeting the cyto 5′-tRNA^AspGUC half and miR-16. The expected terminal structures of the targeted RNA in the treated total RNA samples are shown on the right. In the analyses of cyto 5′-tRNA^AspGUC, the band was similarly shifted up by the CIP treatment and by the T4 PNK treatment, and further upshift was observed by the HCl + CIP treatment, indicating the presence of a 5′-P and a 3′-cP. In the control analyses of miR-16, as expected, the CIP and HCl + CIP treatments identically shifted the band up, whereas the T4 PNK treatment caused no shift, indicating the presence of a 5′-P and a 3′-OH. A dotted red line indicated the position of the main band in NT sample, which helps to show band mobility differences.

MATERIALS

-REAGENTS

• TRisure (Bioline, cat. no. BIO-38032) CAUTION! TRisure is toxic on inhalation. Use a hood, protective clothing, eye protection, and gloves.

• Chloroform (Sigma-Aldrich, cat. no. C2432) CAUTION! Chloroform is toxic on inhalation. Use a hood, protective clothing, eye protection, and gloves.

• Isopropanol (Sigma-Aldrich, cat. no. I9516)

• Ethyl alcohol (Sigma-Aldrich, cat. no. E7023)

• Certified Nuclease-Free Water (BioExpress, cat. no. G-3250)

• Urea (Sigma-Aldrich, cat. no. U5378)

• TBE, 10× LIQUID CONCENTRATE (amresco, cat. no. 0658)

• 40% Acrylamide/Bis Solution, 19:1 (BIO-RAD, cat. no. 1610144EDU) CAUTION! Monomeric acrylamide is a neurotoxin. Use a hood, protective clothing, eye protection, and gloves.

• Ammonium persulfate (APS; Sigma-Aldrich, cat. no. A3678)

• UltraPure TEMED (Life Technologies, cat. no. 15524-010) CAUTION! TEMED is toxic on inhalation. Use a hood, protective clothing, eye protection, and gloves.

• Bromophenol Blue (Sigma-Aldrich, cat. no. B0126)

• Xylene Cyanol FF (Sigma-Aldrich, cat. no. X4126)

• Low Molecular Weight Marker, 10−100 nt (Affymetrix, cat. no. 76410)

• SYBR® Gold Nucleic Acid Gel Stain (Life Technologies, cat. no. S-11494) CAUTION! SYBR Gold is a DNA-binding agent, and thus it is potentially mutagenic. Use protective clothing, eye protection, and gloves.

• Sodium Acetate (3 M), pH 5.5 (Life Technologies, cat. no. AM9740)

• EDTA (0.5 M), pH 8.0 (Life Technologies, cat. no. AM9260G)

• SDS, 20% Solution (Life Technologies, cat. no. AM9820)

• Alkaline Phosphatase, Calf Intestinal (CIP; New England Biolabs Inc, cat. no. M0290)

• 10× CutSmart Buffer (supplied with CIP)

• RNasin® Ribonuclease Inhibitor (Promega, cat. no. N2111)

• Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1) (Life Technologies, cat. no. AM9720) CAUTION! Phenol and chloroform are toxic on inhalation. Use a hood, protective clothing, eye protection, and gloves.

• Linear Acrylamide (Life Technologies, cat. no. AM9520)

• Sodium periodate (Sigma-Aldrich, cat. no. 311448)

• T4 Polynucleotide Kinase (T4 PNK; New England Biolabs Inc, cat. no. M0201)

• 10× T4 Polynucleotide Kinase Reaction Buffer (supplied with T4 PNK)

• Adenosine 5′-Triphosphate (ATP) (New England Biolabs Inc, cat. no. P0756)

• TruSeq Small RNA Library Preparation Kits (Illumina, cat. no. RS-200-0012), containing RNA 3′ Adapter, Ligation Buffer, Stop Solution, RNA 5′ Adapter, 10 mM ATP, T4 RNA Ligase, 25 mM dNTP mix, RNA RT Primer, PCR Mix, RNA PCR Primer, RNA PCR Primer Index, and High Resolution Ladder.

• T4 RNA Ligase 2, truncated (New England Biolabs Inc, cat. no. M0242L)

• SuperScript III Reverse Transcriptase (Life Technologies, cat. no. 18080-044) containing 5× First Strand Buffer and 100 mM DTT.

• RNaseOut (Life Technologies, cat. no. 10777-019)

• TAE, 50× (amresco, cat. no. K915)

• phiX174 DNA-Hae III Digest (New England Biolab, Inc., cat, no. N3026S)

• 6× loading dye (Affymetryx, cat. no. 76715)

• Hydrochloric acid (Sigma-Aldrich, cat. no. H1758) CAUTION! Hydrochloric acid is toxic on inhalation and cause eye and skin burn. Use a hood, protective clothing, eye protection, and gloves. Do not store in a metal container.

• ATP, [γ-³²P]-6000Ci/mmol 10mCi/ml , 250 µCi (Perkin Elmer, cat. no. BLU002Z250UC) CAUTION! All radioisotopes should be used in strict accordance with the regulations and guidelines of one’s institution. ³²P is a high-energy beta emitter. All steps should be conducted behind Plexiglas shielding. Use protective clothing, eye protection, and gloves.

• illustra MicroSpin G-25 Columns (GE Healthcare, cat. no. 27–5325-01)

• PerfectHyb™ Plus Hybridization Buffer (Sigma-Aldrich, cat. no. H7033)

• Extra Thick Blot Filter Paper, Precut, 19 × 18.5 cm (BIO-RAD, cat. no. 1703969)

• SSC BUFFER, 20× LIQUID CONCENTRATE (amresco, cat. no. 0804)

• DMEM, high glucose (Life Technologies, cat. no. 11965-092)

• Fetal Bovine Serum-Optima, Heat Inactivated (Atlanta Biologicals, cat. no. S12450H)

• Penicillin-Streptomycin (Life Technologies, cat no. 15140-122)

• BT-474 cell line (American Type Culture Collection, cat. no. HTB-20) CAUTION! The BT-474 cells were cultured in DMEM containing 10% FBS and penicillin-streptomycin. The absence of Mycoplasma was regularly checked every month.

• C57BL/6J mice (The Jackson Laboratory, stock no. 000664) CAUTION! Mice were treated under appropriate protocols according to relevant Institutional and National regulations. Spleens were obtained from male adult mice at approximately 3-months ages.

EQUIPMENT

• RNase-Free Microcentrifuge tube, 1.7 ml

• RNase-Free Microcentrifuge tube, 0.6 ml

• RNase-Free Round-bottom, Microcentrifuge tube, 2.0 ml

• RNase-Free Microcentrifuge tube, 0.2 ml for PCR

• Vortex

• Filter tips

• Disposable pipettes

• Pipet-Aid (e.g., Corning)

• Thermoblock with shaking (Eppendorf Thermomixer R)

• Corning® Costar® Spin-X® centrifuge tube filters, 0.45 µm pore size (Sigma-Aldrich, cat. no. CLS8162)

• Spectrometers (e.g., TECAN, infinite 200 Pro series)

• Vaccum Filter Unit; 0.22 µm pore size (e.g., BioExpress, cat. no. F-2980)

• Refrigerated benchtop centrifuge (Eppendorf, cat. no. 5424R)

• SE410 Tall Air-Cooled Vertical Electrophoresis Unit (Hoefer, cat. no. SE410)

• SE400 Air-Cooled Vertical Electrophoresis Unit (Hoefer, cat. no. SE400)

• Mini-PROTEAN® Tetra Cell Casting Stand with Clamp Kit for Single Core System (BIO-RAD, cat. no. 1658052)

• Mini-PROTEAN® Tetra Electrode Assembly (BIO-RAD, cat. no. 1658037)

• Buffer Tank and Lid (BIO-RAD, cat. no. 1658040)

• Short Plates (BIO-RAD, cat. no. 1653308)

• Spacer Plates with 1.0 mm Integrated Spacers (BIO-RAD, cat. no. 1653311)

• Mini-PROTEAN® Comb, 10-well, 1.0 mm, 44 µl (BIO-RAD, cat. no. 1653359)

• Power Supply unit

• Gel imaging system (e.g., BIO-RAD, ChemiDoc, cat. no. 1708280)

• Blue light transilluminator (e.g., SYNGENE UltraSlim-LED)

• Vacuum Concentrator (Eppendorf Vacufuge plus, cat. no. 2231000079)

• Thermal Cycler (e.g., Labnet MultiGene™ OptiMax Thermal Cycler, cat. no. TC9610)

• Blade

• 21G Needle

• Crosslinker, shortwave UV (UVP, cat. no. 95–0174-01)

• Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell (BIO-RAD, cat. no. 1703940)

• Liquid scintillation and gamma counters (Hidex, Triathler)

• Amersham Hybond-N+ (GE Healthcare, cat. no. RPN303B)

• Deluxe ProBlot™ Hybridization Systems (Labnet)

• Hybridization Bottles

• Wrap

• Imaging Screen-K (Kodak) (BIO-RAD, cat. no. 1707843)

• Exposure Cassette-K (BIO-RAD, cat. no. 1707861)

• Phosphorimager (e.g., GE Healthcare, Typhoon FLA 7000)

REAGENT SETUP

• 15% acrylamide gel solution containing 7 M urea

  Reagent	Volume/ amount
  Acrylamide: bisacrylamide (19:1), 40% (w/v)	187.5 ml
  TBE, 10×	50 ml
  Urea	210.21 g
  MilliQ water	Up to 500 ml
  Total volume	500 ml

• After filtration using a vacuum filter unit, the solution can be stored at 4 °C for up to 3 months with shielding from light.

• CRITICAL; Change the acrylamide concentration depending on the targeted RNA length.

• 2× loading buffer

  Reagent	Final concentration
  Urea	9 M
  Xylene cyanol	0.05%
  Bromophenol blue	0.05%

• RNA elution buffer

  Reagent	Volume	Final concentration
  SDS solution, 20%	50 µl	0.10%
  EDTA, 0.5 M, pH 8.0	2 µl	0.1 mM
  Nuclease-free water	Up to 10 ml
  Total volume	10 ml

• 8% acrylamide gel solution for native PAGE

  Reagent	Volume
  Acrylamide: bisacrylamide (19:1), 40%	4 ml
  TAE, 50×	400 µl
  MilliQ water	Up to 20 ml
  Total volume	20 ml

• CRITICAL; Change the acrylamide concentration depending on the targeted cDNA length.

PROCEDURE

RNA extraction; TIMING 1 h

• 1.Suspend cells or tissues in 1 ml of TRisure (the volume depends on the sample amount), add 200 µl of chloroform, and vortex for 15 s.
• 2.After incubation at room temperature (20−25 °C) for 3 min, centrifuge at >15000 g for 15 min, 4 °C.
• 3.Transfer the aqueous phase to a new tube.
• 4.Add 500 µl of isopropanol, vortex well, and incubate for 10 min at −20 °C.
• 5.Centrifuge at >15,000 g for 30 min, 4 °C.
• 6.Remove the supernatant and discard, without dislodging the pellet.
• 7.Add 1 ml of 70% ethanol and centrifuge at >7,500 g for 10 min, 4 °C.
• 8.Remove the supernatant completely and discard, without dislodging the pellet.
• 9.Evaporate residual supernatant from pellet for ~45 s at room temperature using a centrifuge vacuum concentrator.
• 10.Dissolve the pellet in an appropriate amount (expected resultant concentration: 1−10 µg/µl) of nuclease-free water.

  CRITICAL STEP; To avoid pellet insolubility, do not dry the pellet for a long time and then immediately add nuclease-free water. Perform pipetting over 100 times to completely dissolve the pellet. Do not dissolve by vortexing and tapping.

  CRITICAL STEP; We recommend to use fresh RNA samples for cP-RNA-seq. Do not use old RNA samples stored in a freezer for a long time, because cP is not very stable in aqueous solution and susceptible to background hydrolysis.

Gel-purification of RNAs with targeted lengths; TIMING 2 d

• 11.Assemble a gel apparatus (e.g., Mini-PROTEAN®, 7 cm × 10 cm; BIO-RAD).
• 12.Add 75 µl of 10% (wt/vol) APS solution and 5 µl of UltraPure TEMED per 10 ml of the 15% acrylamide gel solution containing 7 M urea (See Reagent Setup), gently mix, and then immediately pour into the gel apparatus. To polymerize, incubate for 1−2 h at room temperature.
• 13.After pre-running for 30 min at 200 V, wash the wells using a 21-G needle with a syringe, and then apply the extracted total RNAs from Step 10 mixed with the same volume of the 2× loading buffer.

  CRITICAL STEP; The total RNA amount depends on the amount of targeted RNA in the sample. The amount of targeted RNAs could be estimated by Northern blots. In our 5′-SHOT-RNA analyses, 5′-SHOT-RNA^AspGUC and 5′-SHOT-RNA^HisGUG were clearly detected by Northern blot using 5 µg of BT-474 total RNA ⁵. We loaded 50–100 µg of BT-474 total RNA for this procedure.

• 14.Run the gel at 200 V for 1 h until bromophenol blue reaches the bottom of the gel.

  CRITICAL STEP; Change the electrophoresis time depending on the targeted RNA length. The above condition is appropriate for 20–50 nt targeted RNAs. If the targets have longer lengths such as 50–100 nt, increase the running time and run the gel until xylene cyanol reaches the bottom of the gel.

• 15.Disassemble the apparatus and stain the gel with 0.01% (vol/vol) of SYBR Gold in 1×TBE for 15 min.
• 16.Visualize the gel on a LED blue light transilluminator and then cut the gel region containing the RNAs with the targeted lengths.
• 17.Make a gel breaker tube by puncturing the bottom (3–4 places) of a 0.6-ml microcentrifuge tube with a 21-G needle, and then place the tube into a round-bottom 2.0-ml microcentrifuge tube.
• 18.Place the cut gel fractions into the gel breaker tube and break the gel by centrifuging at >20,000 g for 5 min, 4 °C.
• 19.Add 300–800 µl of the RNA elution buffer (see Reagent Setup) into the produced gel debris in the 2.0-ml microcentrifuge tube.

  CRITICAL STEP; Change the volume of elution buffer depending on the volume of gel debris. The volume of elution buffer should be ~3 times more than that of gel debris.

• 20.Rotate or shake the tube overnight at 4 °C.

  CRITICAL STEP; Confirm that the debris is floating in the buffer and not sticking at the bottom of the tube during rotation.

• 21.Centrifuge at >15,000 g for 5 min, 4 °C and transfer the eluate into a centrifuge tube filter with 0.45 µm pore size.
• 22.Centrifuge at 600 g for 2 min, 4 °C and collect eluate for complete removal of the gel debris.
• 23.Add 10% volume of 3M sodium acetate and 1–2 µl of linear acrylamide and vortex. Next, add one volume of isopropanol, vortex the mixture well, and incubate for 30 min at −20 °C.
• 24.Centrifuge at >15,000 g for 30 min, 4 °C and remove supernatant and discard, without dislodging the pellet.

  CRITICAL STEP; After terminating the centrifugation, remove the supernatant as quickly as possible without losing the pellet, which might only be faint white.

  ?TROUBLESHOOTING

• 25.Add 1 ml of 70% ethanol, followed by centrifuging at 7500 g for 5 min, 4 °C.
• 26.Remove the ethanol completely and dry the sample for 45 s at room temperature using a vacuum centrifuge concentrator.

  CRITICAL STEP; Remove the supernatant as quickly as possible and do not dry the pellet for a long time.

• 27.Dissolve the pellet in nuclease-free water; the amount is dependent on the expected amount of targeted RNA. In our 5′-SHOT-RNA identification, we dissolved the pellet in 50 µl of water.

Phosphatase (CIP) treatment; TIMING 2 h

• 28.
  Add the following 30 µl of reaction mixture into 20 µl of the gel-purified RNA from Step 27, and incubate for 40 min at 37 °C.

  Reagent	Volume (µl) per reaction	Final concentration
  CutSmart buffer, 10×	5	1×
  RNasin	0.5	0.2–0.4 U/µl
  CIP	1	0.2 U/µl
  Nuclease-free water	23.5
  Total volume	30

• 29.Add 50 µl of nuclease-free water and 100 µl of acid phenol-chloroform.
• 30.Vortex the mixture for 10 s and centrifuge at >15,000 g for 5 min, room temperature.
• 31.Transfer the upper aqueous phase into a new tube.

  CRITICAL STEP; Completely avoid contamination from the lower organic fraction.

• 32.Add 200 µl of nuclease-free water, 1 µl of linear acrylamide, 30 µl of 3 M sodium acetate, and vortex the mixture.
• 33.Add 750 µl of ethanol, vortex the mixture, and place the tube for 30 min at −80 °C.
• 34.Centrifuge at 15,000 g for 30 min, 4 °C.
• 35.Remove the supernatant completely and discard, without dislodging the pellet and dry the sample for 45 s at room temperature using a vacuum centrifuge concentrator.

  CRITICAL STEP; Remove the supernatant as quickly as possible and do not dry the pellet for a long time.

• 36.Dissolve the pellet in 45 µl of nuclease-free water.

Periodate (NaIO₄) treatment; TIMING 2 h

• 37.Prepare 100 mM NaIO₄ solution by dissolving in nuclease-free water.

  CRITICAL STEP; Prepare a fresh solution for every experiment. NaIO₄ is light sensitive: therefore, the solution and the following reaction mixture containing NaIO₄ should not be exposed to light (e.g., cover the tube with aluminum foil).

• 38.Add 5 µl of 100 mM NaIO₄ solution into 45 µl of the CIP-treated RNA from Step 36 (final concentration of NaIO₄ is 10 mM), and incubate for 40 min on ice in the dark.
• 39.Add 250 µl of nuclease-free water and perform ethanol precipitation as described in Steps 32–35.
• 40.Dissolve the pellet in 10 µl of nuclease-free water.

  PAUSE POINT; The dissolved RNA can be stored at −80 °C for up to 3 months.

Kinase (T4 PNK) treatment; TIMING 2 h

• 41.
  Add 20 µl of the following T4 PNK reaction mixture into 10 µl of the NaIO₄-treated RNA from Step 40, and incubate for 40 min at 37 °C.

  Reagent	Volume (µl) per reaction	Final concentration
  T4 PNK reaction buffer, 10×	3	1×
  ATP, 10 mM	0.5	166 µM
  RNasin	0.5	0.3–0.6 U/µl
  T4 PNK	1	0.33 U/µl
  Nuclease-free water	15
  Total volume	20

• 42.Add 70 µl of nuclease-free water and perform phenol chloroform extraction and ethanol precipitation as described in Steps 29–35.
• 43.Dissolve the pellet in 10 µl of nuclease-free water.

  PAUSE POINT; The dissolved RNA can be stored at −80 °C for up to 3 months.

Adapter ligation using Illumina TruSeq Small RNA Preparation Kit; TIMING 3 d

• 44.Add 1 µl of 3′-adapter (RA3) into 5 µl of the T4 PNK-treated RNA from Step 43.
• 45.Incubate for 2 min at 70 °C and then immediately place the tube on ice and incubate for 1 min.
• 46.
  Add 4 µl of the following mixture.

  Reagent	Volume (µl) per reaction
  Ligation buffer (HML)	2
  RNase inhibitor	1
  T4 RNA ligase 2, truncated	1
  Total volume	4

• 47.Incubate for 1 h at 28 °C and then overnight at 4 °C.
• 48.Add 1 µl of the stop buffer (STP), incubate for 15 min at 28 °C, and then place the tube on ice.
• 49.Put 1.1 µl/sample of 5′-adapter (RA5) into a new tube, incubate for 2 min at 70 °C, and then immediately place the tube on ice and incubate for 1 min.
• 50.Add 1.1 µl/sample of 10 mM ATP into the tube and mix by pipetting.
• 51.Add 1.1 µl/sample of T4 RNA ligase and mix by pipetting.
• 52.Add 3 µl of the mixture of 5′-adapter, ATP, and T4 RNA ligase from Step 51 into the 3′-adapter-ligated sample from Step 48.
• 53.Incubate for 1 h at 28 °C and then overnight at 4 °C.

  PAUSE POINT; The dissolved RNA can be stored at −80 °C for up to 3 months.

RT-PCR; TIMING 2 h

• 54.In a 0.2 ml PCR tube, add 1 µl of RT primer (RTP) to 6 µl of the adapter ligated-RNA from Step 53.
• 55.Incubate for 2 min at 70 °C in a thermal cycler and then immediately place the tube on ice.
• 56.
  Add 5.5 µl of the following RT reaction mixture and gently mix by pipetting.

  Reagent	Volume (µl) per reaction
  First strand buffer, 5×	2
  dNTP mix, 12.5 mM	0.5
  DTT, 100 mM	1
  RNase inhibitor	1
  SuperScript III reverse transcriptase	1
  Total volume	5.5

• 57.Incubate for 60 min at 50 °C in a thermal cycler.

  PAUSE POINT; The synthesized cDNA can be stored at 4 °C for up to 1 week.

• 58.
  Add 37.5 µl of the following PCR reaction mixture.

  Reagent	Volume (µl) per reaction
  Ultra pure water	8.5
  PCR mMix (PML)	25
  RNA PCR primer (RP1)	2
  RNA PCR primer index (RPIX)	2

  CRITICAL STEP; Use the different RPIX for multiplex sequencing, which can sequence multiple samples simultaneously in a single sequencing lane. The barcode sequences in the primer index will be distinguished and sorted during data analysis.

• 59.
  Amplify the cDNAs in a thermal cycler using the following PCR cycle conditions;

  Step	Temperature (°C)	Time		Number of cycles
  Initial denaturation	98	30 s		1
  Denaturation	98	10 s		11–15
  Annealing	60	30 s
  Extension	72	15 s
  Final extension	72	10 min		1
  Store	4	∞		1

Gel-purification of the amplified cDNAs; TIMING 2 d

• 60.Assemble a gel cast apparatus (e.g., SE400 Air-Cooled Vertical Electrophoresis Unit for 18 cm × 16 cm × 0.75 mm gel; Hoefer).
• 61.Add 150 µl of 10 % (wt/vol) APS solution and 10 µl of UltraPure TEMED per 20 ml of the 8% acrylamide gel solution, gently mix, and then immediately pour into the gel apparatus. To polymerize, incubate for 1–2 h at room temperature.
• 62.Add 6× loading dye to the amplified cDNA samples from Step 59 and apply to the gel. As size markers, also apply a high-resolution ladder and phiX174 DNA-Hae III digest marker.
• 63.Run the gel at 200 V for ~3 h until the xylene cyanol has moved 75% from the top of the gel.

  CRITICAL STEP; Change the electrophoresis time depending on the targeted cDNA length. The above condition is appropriate for 20–50 nt targeted cDNAs. If the target lengths are longer such as 50–100 nt, run the gel until xylene cyanol has moved to the bottom of the gel.

• 64.Disassemble the apparatus and stain the gel with 0.01% (vol/vol) of SYBR Gold in 1×TAE for 15 min.
• 65.Capture the gel image of strained cDNAs using a gel-imaging system (e.g., ChemiDoc; BIO-RAD).

  CRITICAL STEP; Focus on specific cDNA bands that are only present in a lane from a full reaction with T4 PNK treatment and absent in a lane from a negative control reaction without CIP/T4 PNK treatment.

  ?TROUBLESHOOTING

• 66.Visualize the gel on a LED blue light transilluminator and cut the targeted cDNA bands.
• 67.Break the gel as described in Steps 17–18.

  PAUSE POINT; The cut or broken gel can be stored at −20 °C for up to 3 months.

• 68.Add 300–800 µl of nuclease-free water into the gel debris.

  CRITICAL STEP; Change the volume of water depending on the volume of gel debris.

• 69.Rotate or shake the tube overnight at room temperature.

  CRITICAL STEP; Confirm that the debris is floating in the buffer and is not sticking at the bottom of the tube during rotation.

• 70.Take the eluate and perform ethanol precipitation as described in Steps 21−26.
• 71.Dissolve the pellet in 10 µl of nuclease-free water.
• 72.Perform a quality check, Illumina next generation sequencing, and bioinformatics analyses. The analyses of 5′-SHOT-RNAs are described in Honda et al. ⁵.

Biochemical validation of the presence of cP in the identified RNAs: Acid (HCl) treatment; TIMING 4 h

• 73.Incubate the total RNA (1–20 µg) from Step 10 in the following HCl solution for 3 h on ice.
  CRITICAL STEP; To prevent RNA damage from the concentrated acid, mix the total RNA and water first, then add 100 mM HCl solution onto the inner wall of the tube, and then mix by vortexing.

  Reagent	Volume (µl) per reaction	Final concentration
  Total RNA
  HCl, 100 mM	5	10 mM
  Nuclease-free water	Up to 50 µl
  Total volume	50

• 74.Add 250 µl of nuclease-free water and 30 µl of 3 M sodium acetate (pH 5.5) and vortex well. Then add 750 µl of 100% ethanol, vortex well, and incubate for 30 min at −80 °C.
• 75.Centrifuge at >15,000 g for 30 min, 4 °C.
• 76.Remove the supernatant completely and discard and dry the pellet for 45 s at room temperature using a centrifuge vacuum concentrator.
• 77.Dissolve the pellet in an appropriate amount (expected resultant concentration: 1−10 µg/µl) of nuclease-free water and measure the concentration using a spectrometer (e.g., Tecan, infinite 200 Pro series).

  CRITICAL STEP; To avoid pellet insolubility, do not dry the pellet for a long time and immediately add nuclease-free water.

  PAUSE POINT; The dissolved RNA can be stored at −80 °C for up to 3 months.

Phosphatase (CIP) treatment; TIMING 2 h

• 78.
  Incubate a sample of the acid treated (from Step 77) and a sample of non-treated total RNA (1–20 µg) from Step 10 in the following CIP reaction mixture for 40 min at 37 °C.

  Reagent	Volume (µl) per reaction	Final concentration
  Total RNA
  CutSmart buffer, 10×	5	1×
  RNasin	0.5	0.2–0.4 U/µl
  CIP	1	0.2 U/µl
  Nuclease-free water	Up to 50 µl
  Total volume	50

• 79.Add 50 µl of nuclease-free water and perform phenol chloroform extraction and ethanol precipitation as described in Steps 30–36 (addition of linear acrylamide is not required).
• 80.Dissolve the pellet in an appropriate amount (expected resultant concentration: 1−10 µg/µl) of nuclease-free water and measure the concentration by a spectrophotometer. These samples, using acid treated or non-treated total RNA are referred to as “HCl + CIP” or “CIP” samples, respectively.

  PAUSE POINT; The dissolved RNA can be stored at −80 °C for up to 3 months.

Kinase (T4 PNK) treatment; TIMING 2 h

• 81.
  Incubate total RNA (1–20 µg) from Step 10 in the following T4 PNK reaction mixture for 40 min at 37 °C.

  Reagent	Volume (µl) per reaction	Final concentration
  Total RNA
  T4 PNK reaction buffer, 10×	3	1×
  RNasin	0.5	0.3–0.6 U/µl
  T4 PNK	1	0.3 U/µl
  Nuclease-free water	Up to 30 µl
  Total volume	30

• 82.Add 70 µl of nuclease-free water and perform phenol-chloroform extraction and ethanol precipitation as described in Steps 29–35 (addition of linear acrylamide is not required).
• 83.Dissolve the pellet in an appropriate amount (expected resultant concentration: 1−10 µg/µl) of nuclease-free water and measure the concentration by a spectrophotometer. This is referred to as a “T4 PNK” sample.

  PAUSE POINT; The dissolved RNA can be stored at −80 °C for up to 3 months.

Northern blot; TIMING 3 d

• 84.For micro RNA (~22 nt) or 5′-SHOT-RNAs (~34 nt), make 18 cm × 24 cm × 0.75 mm 15% denaturing acrylamide gel (e.g., using SE410 Tall Air-Cooled Vertical Electrophoresis Unit) as described in Steps 11–12.
• 85.
  Radio-label low molecular single nucleotide marker (e.g., Low Molecular Weight Marker, 10–100nt; Affymetrix) by incubating the following reaction mixture for 1 h at 37 °C.

  Reagent	Volume (µl) per reaction
  Low Molecular Weight Marker	1
  32P-γ-ATP, 6,000 Ci/mmol	1
  T4 PNK reaction buffer, 10×	1
  T4 PNK	0.5
  Nuclease-free water	Up to 10 µl
  Total volume	10

• 86.Purify the labeled marker using illustra Microspin G-25 columns. Count the CPM of the marker solution using a liquid scintillation counter (5,000–10,000 CPM of the labeled marker should be applied in the following step).

  CAUTION! Use all radioisotopes in strict accordance with the regulations and guidelines of one’s institution.

• 87.After pre-running the gel from Step 84 for 30 min at 300 V, wash the wells using a 21-G needle with a syringe, and apply the RNA samples [labeled marker (from Step 86), non-treatment total RNA (from Step 10), and three treated total RNAs, “CIP”, “HCl + CIP”, and “T4 PNK” (from Steps 80 and 83)] mixed with the same volume of the 2× loading buffer.
• 88.Run the gel at 300 V for 4–5 h in 1×TBE buffer until the xylene cyanol has moved 75% from the top of the gel.

  CRITICAL STEP; Do not increase the voltage too much to avoid curving of the bands. Change the electrophoresis time depending on the targeted RNA length. The above condition is appropriate for 20–40 nt targeted RNAs. Xylene cyanol matches ~30 nt RNAs in the 15% denaturing acrylamide gel. If targeted RNAs are longer, prepare a larger gel (e.g. using Fisher Scientific; SEQUENCING SYSTEM ALUMINUM 2, cat no. OW S4S) and increase the running time.

• 89.Disassemble the apparatus and transfer the RNAs in the gel to Hybond-N+ membrane using a semi-dry blotting system for 30 min at 400 mA in 1×TBE.
• 90.After drying the membrane, perform UV cross-linking at 120 mJ/cm² twice using UV cross-linker.
• 91.For pre-hybridization, rotate and incubate the membrane with PerfectHyb plus hybridization buffer in hybridization bottle for 1 h at 37 °C.
• 92.
  Prepare ³²P-labeled probe by incubating the following mixture for 1 h at 37 °C, and then purify the labeled probe using illustra MicroSpin G-25 columns. Count the CPM of the probe using a liquid scintillation counter.

  Reagent	Volume (µl) per reaction
  DNA probe, 2 µM	2
  32P-γ-ATP, 6,000 Ci/mmol	1
  T4 PNK reaction buffer, 10×	1
  T4 PNK	0.5
  Nuclease-free water	Up to 10 µl
  Total volume	10

• 93.Add 1,000,000–2,000,000 CPM of the ³²P-labeled probe to the hybridization bottle and incubate overnight at 37 °C.
• 94.Briefly shake the membrane in 2×SSC buffer, discard the buffer, and then wash the membrane twice by rotating and incubating for 20 min at 37 °C in 2×SSC buffer.
• 95.After drying the membrane, cover with wrap and expose to a phosphor-imaging screen for 1 h–overnight at room temperature in a cassette.
• 96.Scan the image using a Phosphorimager (e.g. Typhoon; GE Healthcare), and observe the band differences between samples.

  CRIICAL STEP; In the case of the 20–35 nt target, removal of a cP or P causes an up-shifted band because the effect of the phosphate charge is more critical than that of the phosphate mass. The balance of the effect between charge and mass will vary depending on the size of target RNAs.

  ?TROUBLESHOOTING

TIMING

[cP-RNA-seq]

• Step 1–10, RNA extraction, 1 h

• Step 11–27, Gel-purification of RNAs with targeted lengths, 2 d

• Step 28–36, Phosphatase (CIP) treatment, 2 h

• Step 37–40, Periodate (NaIO₄) treatment, 2 h

• Step 41–43, Kinase (T4 PNK) treatment, 2 h

• Step 44–53, Adapter ligation using Illumina TruSeq Small RNA Preparation Kits, 3 d

• Step 54–59, RT-PCR, 2 h

• Step 60–72, Gel-purification of the amplified cDNAs, 2 d

  [Biochemical validation of the presence of cP in the identified RNAs]

• Step 73–77, Acid (HCl) treatment, 4 h

• Step 78–80, Phosphatase (CIP) treatment, 2 h

• Step 81–83, Kinase (T4 PNK) treatment, 2 h

• Step 84–96, Northern blot, 3 d

TROUBLESHOOTING

Troubleshooting advice can be found in Table 1.

TABLE 1.

Troubleshooting table.

Step	Problem	Possible reason	Solution
24	The RNA pellet is invisible	Low amount of targeted RNA fraction	Add additional 1 µl of linear acrylamide, vortex and centrifuge again.
65	Appearance of many strong bands in the lane “CIP/T4 PNK–” negative control	Insufficient periodate treatment	Use fresh NaIO4 solution prepared immediately before the treatment.
	Weak band from full procedure of cP-RNA-seq	Insufficient amount of targeted RNA	Increase starting material.
		Insufficient enzymatic activity	Change enzymes. Avoid phenol contamination in the samples by not taking any of the lower organic phase during phenol- chloroform extraction.
		RNA degradation during procedure	Perform experiments with careful attention to RNase contamination in RNase-free environment. Clean pipettes, bench top, tube rack, tube, and so on using RNAseZap reagent. Keep the sample tubes on ice during the experiment.
96	Insufficient shift of the bands	Insufficient electrophoresis length	Use a larger gel and perform electrophoresis for a longer time so that band mobility differences from the presence/absence of cP/P can be clearly observed.
		Targeted RNA is too long	It is difficult to observe the band mobility differences from long targeted RNA. Therefore, try a different method, such as the method using poly-A polymerase 39,40.
		Insufficient enzymatic activity	Change enzymes. Avoid phenol contamination in the samples by not taking any of the lower organic phase during phenol- chloroform extraction.

ANTICIPATED RESULTS

Selective amplification of 5′-tRNA halves using the cP-RNA-seq procedure

As an example, we show the native PAGE result from a cP-RNA-seq procedure applied to mouse spleen total RNA (Fig. 3). As indicated by an arrow, the method successfully amplified ~150–155-bp cDNA products corresponding to the targeted 5′-tRNA halves (~32–37 bp). The absence of a band from the negative control procedure without CIP/T4 PNK treatment suggested that the band is from cP-containing RNAs.

Biochemical validation of the presence of a cP in a mouse 5′-tRNA half

As an example, we show the Northern blot results using enzyme-treated mouse spleen total RNAs to validate the presence of a cP in a 5′-tRNA half derived from cytoplasmic (cyto) tRNA^AspGUC (Fig. 4). NT designates the non-treated sample used as a negative control. The band was shifted up by the CIP treatment, suggesting the presence of a P in the 5′-tRNA half. The T4 PNK treatment similarly shifted the band up, and further upshift was observed by the HCl + CIP treatment, indicating the additional presence of a cP. These results indicate that the 5′-tRNA^AspGUC half contains a 5′-P and a 3′-cP. Detection of mouse microRNA-16 (miR-16) was used as a control. Because miR-16 contains 5′-P and 3′-OH, the CIP and HCl + CIP treatments identically shifted the band up, whereas the T4 PNK treatment caused no shift.