KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Stem loop binding protein | MBLI | RN045P Lot# 001 |
| Iron response protein 1 | SCBT | E-12 lot #H0117 |
| Iron response protein 2 | SCBT | 4G11 lot #F317 |
| Immunoglobulin G (mouse) | Thermo Fisher | Catalog # 02–6502 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| TRIzol Reagent | Invitrogen | Cat# 15596026 |
| NAI-N3 | Synthesized in house | N/A |
| Buffer RLT | Qiagen | cat # 79216 |
| DIBO-biotin | Molecular Probes | Cat# 10130670 |
| D-Biotin | Molecular Probes | Cat# B20656 |
| CircLigase II ssDNA ligase | Epicentre | Cat# CL9025K |
| DMSO | Sigma-Aldrich | Cat# D2650 |
| Dynabeads MyOne streptavidin C1 | Life Technologies | Cat# 65002 |
| Dynabeads M-280 sheep anti-rabbit 10 mg/ml | LifeTech | Cat# 11204D |
| Dynabeads MyOne Silane 40 mg/ml | LifeTech | Cat# 37002D |
| Agencourt AMPure XP beads | Beckman Coulter | Cat# A63881 |
| RNA fragmentation reagents | Ambion | Cat# AM8740 |
| T4 polynucleotide kinase | New England BioLabs | Cat# M0201L |
| FastAP thermosensitive alkaline phosphatase | Thermo Scientific | Ct# EF0651 |
| Proteinase K | New England BioLabs | Cat# P8107S |
| SuperScript III Reverse Transcriptase | LifeTech | Cat# 18080044 |
| Turbo DNase | LifeTech | Cat# AM2239 |
| T4 RNA ligase 1, high concentration | New England BioLabs | Cat# M0437M |
| Phusion high-fidelity PCR master mix | New England BioLabs | Cat# M0531L |
| RiboLock RNase inhibitor | Thermo Scientific | Cat# EO0384 |
| RNase H | Enzymatics | Cat# Y9220L |
| RNase cocktail enzyme mix | Ambion | Cat# AM2286 |
| Q5 PCR Master Mix | New England BioLabs | Cat# M0492L |
| Protease Inhibitor Cocktail III | EMD Millipore | Cat# 539134–1SET |
| Exo-SAP-IT | Affymetrix | Cat# 78201 |
| UltraPure 1 M Tris-HCI buffer, pH 7.5 | Invitrogen | Cat# 15567–027 |
| Tween-20 | Sigma Aldrich | Cat# P1379–500ML |
| Chloroform | Ricca Chemical | Cat# RSOC0020–500C |
| HEPES, 1 M | Life Technologies | Cat# 15630–080 |
| Magnesium chloride, 1 M | Ambion | Cat# AM9530G |
| Sodium chloride, 5 M | Ambion | Cat# AM9759 |
| Buffer RWT | Qiagen, | Cat# 1067933 |
| Nonidet P40 (NP-40) | Roche | Cat# 11332473001 |
| N-Lauroylsarcosine sodium salt solution (20%, for molecular biology) | Sigma Aldrich | Cat# L7414–10ML |
| Deoxycholic acid sodium salt | Fisher Scientific | Cat# BP349–100 |
| eCLIP Lysis Buffer | 50 mM Tris-HCl pH 7.4, 100mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate | N/A |
| eCLIP High Salt Wash Buffer | 50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate |
N/A |
| eCLIP Wash Buffer | 20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% Tween-20, 5 mM NaCl |
N/A |
| eCLIP RLTW Buffer | RLT Buffer, 0.025% Tween-20 | N/A |
| eCLIP PKS Buffer | 100mM Tris-HCl pH 7.4, 50mM NaCl, 10mM EDTA, 0.2% SDS |
N/A |
| 3.3X SHAPE Folding Buffer | 333mM HEPES pH 8.0, 20mM MgCl2, 333mM NaCl | N/A |
| 10X SHAPE FS Buffer | 500mM TrisHCl pH 8.0, 750 mM KCl | N/A |
| Cytosolic liver extract | Life Technologies | Cat# HMCYPL |
| Ammonium iron(III) citrate | Acros Organics | Cat# AC612215000 |
| Deferoxamine mesylate | Sigma Aldrich | Cat# D9533 |
| Dimethyl sulfate | EMD Millipore | Cat# D186309 |
| 2-methylnicotinic acid imidazolide (NAI) | EMD Millipore | Cat# 03–310 |
| Manganese (II) chloride solution 1M | Sigma Aldrich | Cat# M1787 |
| 10X RNA EMSA Binding Buffer | 100mM HEPES (7.3), 200mM KCl, 10mM MgCl2, 10mM DTT |
N/A |
| Glycerol | Thermo Scientific | Cat# 15514011 |
| Power SYBR Green PCR Master Mix | Applied Biosystems | Cat# 4368577 |
| Critical Commercial Assays | ||
| RNA Clean and Concetrator Kit | Zymo Research | Cat# R1019 |
| Small-RNA PAGE Recovery Kit | Zymo Research | Cat# R1070 |
| Direct-zol RNA Miniprep Kit | Zymo Research | Cat# R2050R2051 |
| Poly(A)Purist MAG kit | Ambion | Cat# AM1922 |
| RNA 3’ End Biotinylation Kit | Pierce | Cat# 20160 |
| Chemiluminescent Nucleic Acid Detection Module | Thermo Scientific | Cat# 89880 |
| Deposited Data | ||
| Sequencing data and processed fSHAPE and SHAPE reactivities | This paper | Gene expression omnibus (GEO), GSE149767 |
| Experimental Models: Cell Lines | ||
| K562 | ATCC | CCL-243 |
| HepG2 | ATCC | HB-8065 |
| HeLa | ATCC | CCL-2 |
| Oligonucleotides | ||
| /5rApp/ rArGrArUrCrGrGrArArGrArGrCrGrGrUrUrCrArG /3ddC/ | IDT | N/A |
| /5rApp/rArGrArUrCrGrGrArArGrArGrCrGrGrUrUrCrArG/3Biotin/ | IDT | N/A |
| 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA CGCTCTTCCGATCT-3′ | IDT | N/A |
| 5′-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTG CTGAACCGCTCTTCCGATCT-3’ | IDT | N/A |
| /5phos/DDDNNXXXXNNNNAGATCGGAAGAGCGTCGTGGA/iSp18/GGATCC/iSp18/TACTGAACCGC; XXXX=sample barcode | IDT | N/A |
| 5’-rGrUrGrGrArCrGrArCrCrCrCrCrArGrArGrCrGrGrGrGrArGrCrUrGrC-3’ | IDT | N/A |
| 5’- rGrArUrGrGrGrArCrCrUrGrGrCrCrArGrUrGrArCrCrArGrUrCrCrUrCrUrC-3’ | IDT | N/A |
| 5’- rGrCrArCrUrGrGrCrCrUrCrCrArGrArGrCrCrCrGrUrGrGrCrCrArArG-3’ | IDT | N/A |
| 5’- rCrUrGrGrUrUrCrArGrGrCrCrCrArGrArGrGrUrCrCrArArGrCrUrArUrA-3’ | IDT | N/A |
| 5’-rUrCrCrUrGrCrUrUrCrArArCrArGrUrGrCrUrUrGrGrArCrGrGrArArC-3’ | IDT | N/A |
| 5’-AATACGACTCACTATAGGGTATACGAGTAGCTGCGCTCCCCTGCTAGAACCTCCAAACAAGCTCTCAAGGTCCATTTGTAGGAGAACGTAGGGTAGTCAAGCTTCCAACAACAACA-3’ | IDT | N/A |
| Primers for qPCR, see Table S4 | ||
| /5Phos/rArGrArUrCrGrGrArArGrArGrCrArCrArCrGrUrC/3SpC3/ | IDT | N/A |
| 5’-CAGACGTGTGCTCTTCCGA-3’ | N/A | |
| /5Phos/NNNNNNNNNNAGATCGGAAGAGCGTCGTGT/3SpC3/ | IDT | N/A |
| 5’-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’ | IDT | N/A |
| 5’-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3’ | IDT | N/A |
| Software and Algorithms | ||
| Cutadapt 1.14 | (Martin, 2011) | https://cutadapt.readthedocs.io/en/stable/ |
| UMItools 0.5.0 | (Smith et al., 2017) | https://github.com/CGATOxford/UMI-πtools |
| STAR 2.4.0i | (Dobin et al., 2013) | https://github.com/alexdobin/STAR |
| Bedtools 2.25.0 | (Quinlan and Hall, 2010) | https://bedtools.readthedocs.io/en/latest/ |
| Samtools 1.9 | (Li et al., 2009) | https://samtools.github.io |
| RNAfold 2.4.14 | (Lorenz et al., 2011) | https://www.tbi.univie.ac.at/RNA |
| Clipper pipeline | (Van Nostrand et al., 2016) | https://github.com/YeoLab/clipper |
| fSHAPE reactivity software | This paper | github.com/meracorley/fSHAPE |
| fSHAPE- and SHAPE-eCLIP reactivity pipelines | This paper | github.com/meracorley/f-SHAPE-eCLIP |
| Hmmlearn 0.2.1 | Scikit-learn | https://hmmlearn.readthedocs.io/en/latest/ |
| HBPLUS | (McDonald and Thornton, 1994) | https://www.ebi.ac.uk/thornton-srv/software/HBPLUS/ |
| Hbplus_tools | (Corley et al., 2020) | github.com/meracorley/hbplus_tools |
| RNAstructure_tools | This paper | github.com/meracorley/RNAstructure_tools |
| Other | ||
| Detailed protocols | This paper | Methods S1 |