Protocol for acquiring high-quality fresh mouse lung spatial transcriptomics data

Summary

Spatial transcriptomics analysis allows the examination of the biological characteristics and spatial distribution of individual lung cells at a single-cell resolution. However, due to the presence of cavities in the alveoli of the lungs, it is challenging to section them for spatial transcriptomics experiments. Here, we present a protocol for acquiring high-quality fresh mouse lung spatial transcriptomics data. We describe steps for lung perfusion, acquiring frozen slices, collecting cDNA from lung sections, and data analysis.

For complete details on the use and execution of this protocol, please refer to Jiang et al.¹

Graphical abstract

Highlights

• •Steps for maintaining tissue structure integrity using 50% OCT perfusion fluid
• •Instructions on tissue embedding customized for spatial transcriptomics experiments
• •Guidance on slicing procedures adapted for spatial transcriptomics experiments
• •Details for applying spatial transcriptomics technology to lung organs

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Spatial transcriptomics analysis allows the examination of the biological characteristics and spatial distribution of individual lung cells at a single-cell resolution. However, due to the presence of cavities in the alveoli of the lungs, it is challenging to section them for spatial transcriptomics experiments. Here, we present a protocol for acquiring high-quality fresh mouse lung spatial transcriptomics data. We describe steps for lung perfusion, acquiring frozen slices, collecting cDNA from lung sections, and data analysis.

Before you begin

The lungs are vital organs for performing essential physiological functions such as gas exchange and metabolism, largely mediated by the intricate three-dimensional structures known as alveoli. The arrangement of various cell types, including AT1, AT2, and stromal cells, within these structures plays a crucial role in lung development, injury and regeneration, and the onset of diseases. Understanding the spatial and temporal characteristics and functions of these cells is, therefore, critical for gaining a comprehensive understanding of lung biology. The emergence of spatial transcriptomics technologies, such as Visium (10× Genomics) and Stereo-seq (Chen et al.),² has made it possible to detect these aspects. However, obtaining complete lung tissue sections is challenging due to alveolar cavities, which pose a significant barrier to applying of spatial transcriptomics technologies in lung research. To overcome this challenge and address the early-stage application obstacles, a protocol based on Stereo-seq (Chen et al.)² has been developed that provides practical solutions for lung tissue pre-treatment and spatiotemporal experiments, enabling the application of spatial transcriptomics in the field of lung research.

Institutional permissions

The animal study was reviewed and approved by the Ethics Committee of BGI (Permit No.20220816694).

Note: Experiments on animals or humans must be carried out only after obtaining relevant ethical approval.

Prepare equipment

Timing: 30 min

• 1.
  Pre-cool cryotome.

  • a.
    Pre-cool the case to −20°C.
  • b.
    Pre-cool the sample head to −20°C.
• 2.
  Pre-heat metal bath.

  • a.
    Set 37°C for permease preheating.
• 3.
  Set fluorescence microscope.

  • a.
    Set up the FITC access in advance.
• 4.Prepare the commercial kits for Stereo-seq technology.

Prepare working solution

Timing: 30 min

• 5.
  0.01 N HCl (store at room temperature (23°C–26°C) no more than one week).

  • a.
    Dilute 3M concentrated HCl with Nuclear Free Water (NF-Water) to 0.1 N to store at room temperature for on more than one month.
  • b.
    Dilute 0.1 N HCl with NF-water to 0.01 N to store at room temperature for no more than one week as working solution, pH accurate to 2.
• 6.
  1 × Permeabilization reagent solution (−20°C).

  • a.
    Dissolve PR Enzyme powder (STOmics) with 1 mL of freshly prepared 0.01 N HCl to 10 × Permeabilization reagent stock solution.

    Note: Mix gently through the pipette, not vortex.

  • b.
    Dilute 10 × Permeabilization reagent stock solution 10 μL–100 μL with 0.01 N HCl to get 1 × Permeabilization reagent solution and store at −20°C.
• 7.
  5 × SSC (room temperature (23°C–26°C)).

  • a.
    Take 5 mL of 20 × SSC and dilute it with 20 mL NF-Water to a final volume of 25 mL.
• 8.
  0.1 × SSC (room temperature (23°C–26°C)).

  • a.
    Take 100 μL 20 × SSC and dilute it with 19.9 mL NF-Water to a final volume of 20 mL.
• 9.
  DNA Clean Beads (4°C).

  • a.
    Remove them from the fridge (4°C) in advance to equilibrate them at room temperature for approximately 30 min.
• 10.
  RT Oligo (−20°C).

  • a.
    Short spin the primer tube, dissolve RT Oligo (STOmics) in 79 μL TE buffer.
  • b.
    Close the lid, vortex the tube for seconds at highest speed and short spin the tube.
• 11.
  Glycerol (room temperature (23°C–26°C)).

  • a.
    Remove it at least 5 min in advance and equilibrate to room temperature before using it.
• 12.
  PR Rinse Buffer (with 5% RI) (−20°C).

  • a.
    Prepare at least 100 μL of Rinse Buffer (95 μL PR Rinse Buffer (STOmics) + 5 μL RI (STOmics)) for each Stereo-seq chip.
  • b.
    Place on ice and equilibrate at room temperature for 5 min before usage.

Note: RI is a part of STOmics kit, and it can be replaced by RNase inhibitor, and the use concentration is 2 U/μL.

• 13.
  50% OCT/PBS (room temperature (23°C–26°C)).

  • a.
    Mix OCT and 1 × PBS with same volume to a final volume of 10 mL for a mouse.
  • b.
    Sit at room temperature for approximately 30 min to minimize bubbles.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Nuclease-free water (NF water)	Ambion	AM9937
Anhydrous ethanol	Xilong Scientific Co., Ltd.	72188-01
AMPure XP	Agencourt	A63882
HCl	Sigma-Aldrich	2104-50ML
Glycerol	Solarbio	G8190
20 × SSC	Ambion	AM9770
Methanol	Sigma	34860-1L-R
Other
Cryotome	Dakewe	CT520
Fluorescence microscope (splicing function)	Stereo-OR 100	SCI-01-016
Metal bath	BIOBASE	BJPX-DB2
Centrifuge	Kylin-Bell	LX-200B
Whirlpool mixer	Kylin-Bell	QL-901
T100 thermal cycler	Bio-Rad∗	1861096
ProFlex 3 × 32-well PCR system	Applied Biosystems∗	4483636
Slide spinner	Labnet	C1303-T
NEBNext magnetic separation rack	NEB	S1515S
DynaMag-2 magnet	Thermo Fisher Scientific	12321D
Qubit 3.0 fluorometer	Thermo Fisher Scientific	Q33216
4200 TapeStation system	Agilent	G2991BA
Water bath-slide dryer	KEDEE	KD-H
Bio-fragment analyzers	BiOptic	Qsep100
Stereo-seq Transcriptomics T Kit	STOmics/BGI	111KT114
Stereo-seq Chip T slide (1 cm∗1 cm)	STOmics/BGI	210CT114
SAKURA Tissue-Tek O.C.T. compound (OCT)	Sakura	4583
RNeasy Mini Kit	QIAGEN	74104
Hematoxylin and eosin (H&E)	Solarbio	G1120
VAHTS DNA clean beads	∗Vazyme	N411-02
Qubit ssDNA Assay Kit	Invitrogen	Q10212
Qubit dsDNA HS Assay Kit	Invitrogen	Q32854
Clear adhesive film	MicroAmp	4306311

Note: All equipment can be replaced by equipment with the same functionality, except for Stereo-seq Transcriptome T Kit and Stereo-seq Chip T Slide (1 cm∗1 cm).

Materials and equipment

Perfusion solution

Reagent	Final concentration	Amount
OCT	0.5 ×	5 mL
PBS	0.5 ×	5 mL
Total	N/A	10 mL

Store at room temperature (23°C–26°C) for up to 1 month

Tissue ssDNA staining solution

Reagent	Final concentration	Amount
5 × SSC	N/A	94.5 μL
Qubit ssDNA Reagent	N/A	0.5 μL
RNase inhibitor	2 U/μL	5 μL
Total	N/A	100 μL

Store at room temperature (23°C–26°C) for up to 1 week

10 × Permeabilization Reagent Stock Solution

Reagent	Final concentration	Amount
PR Enzyme powder (Stereo-seq Transcriptomics T Kit)	10 ×	100 μL
HCl	0.01 M	900 μL
Total	N/A	1 mL

Store at −20°C for up to 3 months

1 × Permeabilization Reagent Solution

Reagent	Final concentration	Amount
10 × Permeabilization Reagent Stock Solution	1 ×	100 μL
HCl	0.01 M	900 μL
Total	N/A	1 mL

Store at −20°C for up to 1 week

RT Mix

Reagent	Final concentration	Amount
RT Reagent	N/A	80 μL
RT Additive	N/A	5 μL
RI	N/A	5 μL
RT Oligo	N/A	5 μL
Reverse T Enzyme	N/A	5 μL
RT Reagent	N/A	80 μL
RT Additive	N/A	5 μL
Total	N/A	100 μL

Store at −20°C for up to 1 week

cDNA Release Mix

Reagent	Final concentration	Amount
cDNA Release Buffer	N/A	380 μL
cDNA Release Enzyme	N/A	20 μL
Total	N/A	400 μL

Store at −20°C for up to 1 week

PCR Mix

Reagent	Final concentration	Amount
cDNA Amplification Mix	N/A	50 μL
cDNA Primer	N/A	8 μL
cDNA	N/A	42 μL
Total	N/A	100 μL

Store at −20°C for up to 1 week

Qubit dsDNA Mix

Reagent	Final concentration	Amount
Invitrogen Qubit dsDNA HS Buffer	N/A	199 μL
Qubit dsDNA HS Reagent 200 ×	1 ×	1 μL
Total	N/A	200 μL

Store at 4°C for up to 1 week

Step-by-step method details

Step one: Pulmonary perfusion

Timing: 1 h

The following steps detail the perfusion and embedding of the mouse lung tissue to get the complete and unbroken more lung sections.

• 1.
  Dissecting Mice.

  • a.
    Mice are sacrificed.
  • b.
    Move mice to a flat experimental table and perform laparotomy immediately.
  • c.
    Expose the heart and insert a needle from the left ventricle.
  • d.
    Inject 1 × PBS slowly for blood replacement until the lungs turn white.

Note: Various methods can be used for euthanizing mice, and there are no significant differences between them. However, it is important to handle the process quickly to avoid wasting time.

• 2.
  Perfusion.

  • a.
    Reveal the trachea and insert the needle from the upper third of the lung. Following insertion, secure the area surrounding the needle with a ligature.
  • b.
    Perfuse the lung in situ with 50% OCT/PBS solution, which fills the internal bubble cavity and replaces any excess fluid. Following perfusion, the lungs gradually expand.
  • c.
    Hold until the lungs are fully inflated, and carefully extract them from the chest cavity.

Note: Complete this step as soon as possible while ensuring the quality of the perfusion, and the next step of frozen embedding should be carried out as soon as possible, in order to ensure the freshness of the sample to prevent RNA degradation.

• 3.
  Frozen embedding.

  • a.
    Select an appropriate size embedding cassette based on the required length of the lung tissues for the study.

    • i.
      Label the cassette with relevant information.
    • ii.
      Pre-coat the bottom of the embedding cassette with room temperature (23°C–26°C) OCT, with a controllable thickness of 2 mm.
    • iii.
      Place it on the cryostat/freezing stage with dry ice for pre-cooling immediately.
  • b.
    Take a clean culture dish and place it at an angle.

    • i.
      Add approximately 4 mL of OCT underneath.
    • ii.
      After the OCT in the embedding cassette has partially solidified, rinse the perfused and non-collapsed mouse lung tissues in the OCT for 2–3 s.
    • iii.
      Place the lung in the embedding cassette pre-coated with OCT according to the previous labeling immediately.
  • c.
    Fill the embedding cassette with bubble-free OCT, submerging the mouse lung completely.
  • d.
    Place the cassette on the cryostat/freezing stage with dry ice for freezing and take photographs for documentation (Figure 1).

Note: It is best to complete within 1 h, if you are particularly skilled, you can ask to complete within 30 min in order to ensure the freshness of the sample to prevent RNA degradation.

Note: During the freezing process, it is crucial to ensure that the mouse lung organs are fully submerged in OCT. If any part of the organs is exposed or the layer of OCT is too thin, additional OCT should be added before complete solidification occurs. Generally, a distance of 2–3 mm above the plane of the embedding box should be maintained for optimal results. The sample can then be placed on a frozen platform (−20°C) until solidified and stored at −80°C for up to one year.

Figure 1.

The frozen embedded mouse lungs after perfusion using 50% OCT/PBS

Step two: Acquisition of frozen slices

Timing: 4 h

The morphological integrity of slices exerts a significant impact on experimental outcomes. It is imperative to employ slices with complete internal architecture to minimize the damage to spatial structure and morphology. And in this step, tissue is sectioned and RNA quality is determined.

• 4.
  Microtome preparation.

  • a.
    Pre-chill the frozen microtome case by setting its temperature to −13°C.
  • b.
    Pre-chill the sample holder to −17°C.
  • c.
    Prepare a brush, razor blade, 1.5 mL centrifuge tube, and tweezers beforehand, and pre-chill these tools in the box.
• 5.Remove the frozen embedded mouse lung tissues from the −80°C freezer and place them in the pre-cooled cryostat chamber at −20°C to equilibrate for 30 min.
• 6.
  Fixing tissue block.

  • a.
    Apply a layer of room temperature (23°C–26°C) OCT uniformly on the sample head, preferably thicker than 2 mm.
  • b.
    Melt some of the ice crystals on the surface of the sample head with a gloved hand and wipe them off with dust-free paper.
  • c.
    Place the embedded sample in the direction of the desired slice image and press gently.
  • d.
    Place it onto the frozen surface and allow for complete freezing and solidification.
  • e.
    Transfer it onto the frozen head of the microtome once fully solidified.
• 7.
  Pruning tissue block.

  • a.
    Repair the tissue block according to the marking information, in order to obtain the desired cross-sectional view of the tissue and fits on the Stereo-seq chip.
  • b.
    To fit on the Stereo-seq chip, the sample section should be smaller than 0.9 cm × 0.9 cm.
  • c.
    Remove the excess OCT around the tissue blocks and retain some OCT blocks for tissue transfer.
• 8.
  Slicing beginning.

  • a.
    Slice the samples with a thickness of 25 μm until the tissue is faintly visible.
  • b.
    Replace it with a slice thickness of 10 μm.
• 9.
  The RNA integrity sequencing.

  • a.
    Cut 20 slices in a row.
  • b.
    Transfer all sections to a 1.5 mL centrifuge tube for subsequent measurements of tissue RNA integrity (RIN).
  • c.
    Extract tissue RNA using the RNeasy Mini Kit and test by 4200 TapeStation System using RNA ScreenTape Analysis (Figure 2).

Note: RNeasy Mini Kit protocol: https://www.qiagen.com/zh-us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-kits

Figure 2.

The RIN of mouse lung tissues is 9.6, greater than 7

RNA ScreenTape Analysis protocol : https://www.agilent.com/zh-cn/product/tapestation-automated-electrophoresis/tapestation-rna-screentape-reagents/rna-screentape-analysis-228268#productdetails

Note: The RNA has a sufficient quality if the RIN score exceeds 7.

• 10.
  Formal patch.

  • a.
    Employ the Cryotome (DAKEWE) to slice the mouse lung into 10 μm sections for academic research purposes.
  • b.
    With two small brushes, delicately flip the sections and then use the brush to expand it without breakage and wrinkles.
  • c.
    Use tweezers to grasp a corner of the Stereo-seq chip, flip it over, and align the tissue area with the section.
  • d.
    Gently press down to ensure complete tissue attachment onto the chip surface before placing it in a 37°C oven (Water Bath-Slide Drier, KEDEE) for 3 min to dry immediately.
  • e.
    Select the adjacent tissue section to attach to the surface of glass slide for H&E staining.

Note: Therefore, one section is for Stereo-seq chip, the adjacent section is for glass slide, and below briefly named Stereo-seq chip and glass slide respectively (Figure 3).

Note: Handle the slice gently with a brush, avoiding direct contact with the tissue.

Figure 3.

The workflow of attachment of chip and section

Step three: Tissue fixation, fluorescence

Timing: 1–2 h

In this step, tissue is fixed, and imaged to determine the cell boundaries for cell segmentation analysis.

• 11.
  Tissue fixation.

  • a.
    Place the dried Stereo-seq chip and the glass slide separately in pre-cooled −20°C methanol, ensuring the tissue is completely submerged.
  • b.
    Fix at −20°C for 30 min.
  • c.
    Use dust-free paper to remove excess methanol from the back and surrounding areas of the Stereo-seq chip.
  • d.
    Place it in a fume hood for 4–6 min to ensure complete dissipation of methanol to eliminate the influence of methanol solution on the subsequent reaction.
• 12.
  Fluorescence photography.

  • a.
    Add 100 μL of tissue ssDNA fluorescent staining solution onto each chip and incubate at room temperature (23°C–26°C) in the dark for 5 min.

    Note: ssDNA fluorescent is a critical step for Stereo-seq experiment, and that is aim to obtain the each cell boundary imaging which are merged with the sequencing visual data to do cell segmentation for single cell analysis.

  • b.
    Wash the chip with 100 μL 0.1 × SSC slowly.
  • c.
    Gently blow-dry the surface of the chip with an air gun, and slowly add glycerol to the center of the tissue.
  • d.
    Cover with a coverslip and immediately take photographs to avoid fluorescence quenching after glycerol has infiltrated the entire chip.
  • e.
    Use a fluorescence microscope to scan the entire chip and take a full ssDNA imaging.
  • f.
    Invert the Stereo-seq chip onto a surface dish and cover the chip completely with 5 × SSC.
  • g.
    Incubate for 8 min to allow for permeation.
  • h.
    After the Stereo-seq chip and coverslip have entirely separated, place the Stereo-seq chip right-side up on dust-free paper.
  • i.
    Absorb the liquid from the back and around the chip.
  • j.
    Wash the surface of the chip with 100 μL 0.1 × SSC slowly (Figure 4).

    Note: The surface of the Stereo-seq chip is coated with nucleic acid groups which can be stained by ssDNA. In order to register with the sequencing results, the Stereo-seq chip was specially treated with grid, that is, there were straight lines with different intervals without fluorophores, so that some dark lines without fluorescence signals could be seen after ssDNA staining (Figure 4 zoom in red arrows). Sequencing visualizations also show low signal formation lines at dark lines due to the absence of nucleic acid groups. The photographed lines are compared with the sequenced lines and the pieces are put together to connect the spatial information.

• 13.H&E staining.
  H&E staining is used for observing the organizational form and structure, as well as pathological features.

  • a.
    Drop 500 μL hematoxylin to the glass slide staining for 2 min to ensure that the dye evenly covers all tissue areas.
  • b.
    Rinse the tissue with tap water at a slow flow for 2 min.
  • c.
    Drop 500 μL of differentiation solution to differentiate the colored tissue for 1 min, ensuring that the differentiation solution evenly covers all tissue areas.
  • d.
    Repeat step b.
  • e.
    Drop about 500 μL eosin to stain for 1 min, ensuring that the dye evenly covers all tissue areas.
  • f.
    Repeat step b.

    Note: When dropping hematoxylin, eosin and differentiation solution, make sure that the liquid covers the tissue area completely to prevent uneven staining or decolorization of the nucleus and cytoplasm. When rinsing with running water, let the water flow to a place far away from the tissues, and to flow slowly over the tissue samples, but do not let the water flow impact on the tissue area directly, to prevent the tissues from being dislodged. For different organ tissue samples with different thicknesses, treatment time and the concentration of differentiation solution can be adjusted accordingly to prevent excessive decolorization or incomplete decolorization.

  • g.
    Put the colored glass slide into the baking machine to dry for 3 min until the moisture on the surface of the glass slide is dried.
  • h.
    Add about 8 μL of glycerol onto the glass slide, cover and seal the glass slide.

    Note: the coverslip should be closed slowly to prevent air bubbles from affecting observation and photo recording.

    Note: If you want to prolong the storage time of the sample glass slide, you can also use alcohol and xylene to treat the glass slide and then seal them with neutral gum to obtain glass slide that can be stored for at least three years.

  • i.
    Record the sample number and name it, indicate the chip number, time, sample name and other important information.
  • j.
    Use the fluorescence microscope.

    • i.
      Select the transmission fluorescence scanning mode.
    • ii.
      Select the TRANS channel, 10 times the lens.
    • iii.
      Scan the whole chip, take a full picture.
    • iv.
      Save the entire folder of photographic results (including the large picture obtained by stitching and every small pictures).

Figure 4.

The ssDNA imaging of mouse lung and zoom in partly

Step four: RNA capturing and cDNA library generation

Timing: 1 h

In this step, tissue is permeabilized to capture RNA, reverse transcription into cDNA and sequence.

• 14.
  Tissue permeabilization.

  • a.
    Slowly drip 100 μL of 1 × Permeabilization Reagent Solution onto the chip surface to completely cover the chip surface.
  • b.
    Slowly transfer to oven at 37°C for 8 min.
  • c.
    Suck up the liquid on the surface of the permeated Stereo-seq chip immediately and clean once using 100 μL 0.1 × SSC.
• 15.
  Reverse transcription.

  • a.
    Drop 100 μL of reverse transcription reagent onto the chip and cover the surface of the chip.
  • b.
    Transfer to an oven at 42°C for reverse transcription reaction for 2 h.
  • c.
    Suck up the liquid on the surface of the reverse-transcribed chip and rinse once using 100 μL 0.1 × SSC, and transfer to a 24-well plate.
• 16.
  Tissue removal.

  • a.
    Add 400 μL of tissue removal reagent to each chip.
  • b.
    Transfer the Stereo-seq chip to the oven at 55°C and incubate for 10 min.
  • c.
    Transfer the Stereo-seq chip into the new well in the same 24-well plate.
  • d.
    Add 1 mL 0.1 × SSC reagent and gently rinse the Stereo-seq chip surface to remove tissue which is attached onto the Stereo-seq surface before, until tissue is invisible to the naked eye.
• 17.
  cDNA release.

  • a.
    Transfer the Stereo-seq chip into the new well.
  • b.
    Add 400 μL of release reagent and cover with the Clear Adhesive Film (STOmics, heat resistant sealing film can be replaced) to completely seal it to prevent liquid evaporation.
  • c.
    Transfer to a 55°C oven for 3 h.
• 18.
  First cDNA Purification and PCR amplification.

  • a.
    Remove the 24-well plate after the reaction, aspirate the liquid from each reaction well, and transfer it to a 1.5 mL Eppendorf (EP) tube.
  • b.
    Wash the reaction wells with 350 μL NF-Water, mix the 400 μL reaction liquid from cDNA release step and wash 350 μL NF-Water to a final 750 μL volume.
  • c.
    Accurately measure the volume (If final volume is less than 750 μL, add NF-Water to make up).
  • d.
    Use DNA clean beads (AMPure XP) at 0.8 × the volume for purification to obtain the cDNA solution.

    • i.
      Mix the collected cDNA (750 μL) with the beads in a ratio of 1:0.8. Vortex the mix.
    • ii.
      Incubate it at room temperature (23°C–26°C) for 10 min.
    • iii.
      Spin down and place the tube onto a magnetic separation rack for 3 min until the liquid becomes clear.
    • iv.
      Carefully remove and discard the supernatant with a pipette (If foams are seen on the cap, discard them with a pipette).
    • v.
      Keep the tube on the magnetic separation rack and add 1 mL of freshly prepared 80% ethanol to wash the beads by rotating the tube on the magnetic rack.
    • vi.
      Incubate for 30 s and carefully remove and discard the supernatant.
    • vii.
      Repeat step 4) one more time.
    • viii.
      Keep the tube on the magnetic rack, and open the lid to air-dry the beads at room temperature (23°C–26°C) until no wetness (reflectiveness) is observed.
    • ix.
      Drying times will vary but will be approximately 5–8 min.
    • x.
      Add 22 μL of NF-water to the dried beads. Mix the beads and nuclease-free water by vortexing.
    • xi.
      Incubate at room temperature (23°C–26°C) for 5 min.
    • xii.
      Spin down briefly and place the sample tube onto a magnetic separation rack for 3–5 min until the liquid becomes clear.
    • xiii.
      Transfer the supernatant (∼21 μL) into a new 0.2 mL EP tube.
    • xiv.
      Add another 22 μL of nuclease-free water to the dried beads in step 7) for a second elution.
    • xv.
      Mix the beads and NF-water by vortexing.
    • xvi.
      Incubate at room temperature (23°C–26°C) for 5 min.
    • xvii.
      Spin down briefly and place the sample tube onto a magnetic separation rack for 3–5 min until the liquid becomes clear.
    • xviii.
      Transfer the supernatant (∼21 μL) into the 0.2 mL EP tube in step 8) and obtain a combined eluted cDNA (∼42 μL).
  • e.
    Prepare a 100 μL PCR reaction system by Table 1 and perform a PCR reaction by Table 2.
• 19.
  Second cDNA purification.

  • a.
    Mix the PCR product (100 μL) with pre-equilibrated magnetic beads at 1:0.6.
  • b.
    Shake well to mix. Incubate at room temperature (23°C–26°C) for 10 min.
  • c.
    After brief centrifugation, place the PCR tube on a magnetic stand and let it stand for 3 min until the liquid is clear.
  • d.
    Remove the supernatant.
  • e.
    Keep the centrifuge tube on the magnetic stand and add 200 μL of 80% ethanol for washing.
  • f.
    Wash the magnetic beads by rotating the centrifuge tube on the magnetic stand.
  • g.
    Let it stand for 30 s, carefully aspirate, and discard the supernatant.
  • h.
    Repeat step c.
  • i.
    Keep the centrifuge tube on the magnetic stand, open the lid, and air dry at room temperature (23°C–26°C) for 5–8 min until the surface of the magnetic beads is free of reflection and cracking.
  • j.
    Resuspend the magnetic beads in 42 μL of TE buffer, shake well, and let it stand at room temperature (23°C–26°C) for 5 min.
  • k.
    Briefly centrifuge and place the tube on a magnetic stand for 3–5 min until the liquid is clear. Transfer the supernatant to a new 1.5 mL centrifuge tube.
  • l.
    Take 1 μL of the cDNA sample and use the Qubit dsDNA HS Kit to measure and record the concentration.
  • m.
    Detect the fragment distribution of cDNA by Qsep100 Bio-Fragment Analyzers using NR2 Standard Cartridge (https://www.bioptic.com.cn/ProductDetail/5133859.html) (Figure 5).

Note: A qualified cDNA sample should have a main fragment distribution peak appearing at around 600- 1,200 bp (Figure 5) by Qsep100, and a yield higher than 20 ng.

Pause point: The purified cDNA sample can be stored at −20°C for up to 3 month.

• 20.Ship the cDNA to China National GeneBank (CNGB) for Stereo-seq sequencing.
  Use the following parameters to perform the sequencing run:

  • a.
    Without sample barcode sequenced (for only one sample): choose paired-ended mode with 50 cycles of Read 1 and 100 cycles of Read 2.
  • b.
    Use dark cycles on Read 1 from 26 to 40 cycles.
  • c.
    With sample barcode sequenced (for two or more samples): choose paired-ended mode with 50 cycles of Read 1 and 100 cycles of Read 2 and an additional 10 cycles of sample barcode.
  • d.
    Use dark cycles on Read 1 from 26 to 40 cycles.

Table 1.

PCR reaction master mix

Reagent	Amount
cDNA Amplification Mix	50 μL
cDNA Primer	8 μL
cDNA	42 μL

Table 2.

PCR cycling conditions

Steps	Temperature	Time	Cycles
Initial Denaturation	95°C	5 min	1
Denaturation	98°C	20 s	15 cycles
Annealing	58°C	20 s
Extension	72°C	3 min
Final extension	72°C	5 min	1
Hold	12°C	forever

Figure 5.

The fragment distribution of cDNA

Expected outcomes

The Stereo-seq dataset analysis work can refer to Chen et al.² for details. Optimal Stereo-seq data with viable lung cells. Please refer to Jiang et al.¹ for details.

Some indicators can be valued about lung dataset.

 The Stereo-seq visualization of sequencing data can be well merged with ssDNA imaging by track line. (Notation in “Fluorescence photography” section)

 There are no obvious lung tissue damage or rupture in Stereo-seq visualization and ssDNA imaging (Figure 6).

Figure 6.

The section statu of meeting the required

 The Stereo-seq sequencing saturation is more than 80%. If less than 80%, you can take additional sequences.

 The gene average number in bin 50 (about 25 μm × 25 μm) is more than 500 which allows you can do the most analysis.

Limitations

The faster the “lung perfusion and embedding process”, the better. The shorter the time, the higher quality of spatial transcriptomics data can be generated. We suggest that it is best to complete within 1 h, if you are particularly skilled, you can ask to complete within 30 min. Although there were still a small number of cavities in the lung after perfusion, internal cavity rupture during biopsy was easily avoided with this optimized protocol for mouse lung tissue. We suggest that the level of Figure 6 is a better tissue section, and Stereo-seq experiments can be carried out. For other types of tissues, for example, other organs with internal cavities, lung organs of other body types. Because of the different structure and size of the organ, the difficulty of perfusion is also different, and further optimization is required for both perfusion protocol and permeabilization time.

In conclusion, the perfusion OCT/PBS scheme is very friendly for conducting Stereo-seq experiments on tissues with cavities. As long as the perfusion is successful, it can basically meet the needs. However, how to perfusion depends on the structural characteristics of different tissues, and this is a practical limitation.

Troubleshooting

Problem 1

The mouse lung contains numerous cavities and water, which renders it susceptible to internal section breakage and bubble structure damage during the sectioning process, ultimately leading to loss of original spatial position and structure which resulting in there are no qualified slices to perform the Stereo-seq experiment, or even if they are carried out, the repeatability will be poor.

Potential solution

Step one: Pulmonary perfusion.

• •The mouse lungs are perfused with a 50% OCT and 50% PBS mixture to eliminate air bubbles within the tissue, thereby achieving a comprehensive section of internal tissue structure while minimizing damage to spatial morphology.

Problem 2

The original experimental design failed to ensure the RNA quality in mouse lung tissue, resulting in poor data quality such as RIN is less than 7, cDNA fragment is less than 600 bp and the gene number in sequencing data is less than 500 in bin50. The steps are including.

• •The sampling and embedding time of mouse lung organs is too long, and exposure to air for more than 3 h will accelerate the degradation of RNA molecules.
• •The storage temperature of the embedded samples is higher than −60°C and the storage time is more than 1 year.
• •Stored samples undergo repeated freezing and thawing.

Potential solution

Step one: Pulmonary perfusion.

• •By incorporating a 5% volume ratio of RNase inhibitor (or RI in STOmics Kit) into the perfusion solution, RNA degradation in lung tissues following air exposure can be mitigated, enhancing the yield of high-quality lung tissues and enabling optimal conditions for conducting spatial transcriptome experiments to obtain superior cDNA.
• •The step one: Pulmonary perfusion, it is best to complete within 1 h, if you are particularly skilled, you can ask to complete within 30 min in order to ensure the freshness of the sample to prevent RNA degradation.
• •The storage temperature of the embedded samples is better in −80°C and the storage time is no more than 1 year.
• •Stored samples don’t undergo repeated freezing and thawing.

Problem 3

How to ensure ssDNA staining is of sufficient quality? The ssDNA imaging is critical for cell segmentation by merged with sequencing data which is described in Step three: point 12.Fluorescence photography. Therefore, obtain the high quality ssDNA imaging is necessary.

Potential solution

Step three: Point 12. Fluorescence photography

We can have the specific criterions by below points using ImageJ System.

• •Open the ssDNA imaging by ImageJ System, and the signal value will be displayed automatically. The fluorescence signal value of the tissue cells was greater than 60, and the boundary of the cells are clear and clearly distinguishable from the background.
• •Adjust the “Brightness” in ImageJ System till the track could be seen. The track lin is clear like the imaging of Figure 4.

Problem 4

If the cDNA amplification product is less than 20 ng, or the fragment distribution is not concentrated in the 600–1200 bp range, there may be a problem with the experimental product, resulting in inability to sequence or poor sequencing quality.

Potential solution

Step three: Point 19. Second cDNA purification.

• •One: the cDNA amplification product is less than 20 ng, but the fragment distribution is concentrated in the 600–1200 bp range. This means that the length of RNA is normal but the total amount is not enough. It is necessary to first check the size of the tissue area attached to the 1 cm ∗ 1 cm Stereo-seq chip. If the tissue is too small, the total amount of RNA is bound to be very small. It is recommended that the tissue area be greater than 6 mm∗6 mm, or paste multiple small tissues on a chip. Secondly, if the tissue is large enough and the total amount of RNA is not enough, it is recommended to perform more PCR cycles, which are generally increased within 10 times at a time.
• •Two: the cDNA amplification product is less than 20 ng, and the fragment distribution is not concentrated in the 600–1200 bp range. This means that the length of RNA is short and the total amount is not enough. I suggest testing the fragment distribution after several rounds of PCR, if it is still short and low, it means that this step has failed. It is necessary to check the RIN value of the sample and the aforementioned experimental process.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the technical contact, Chuanyu Liu (liuchuanyu@genomics.cn).

Technical contact

Technical questions about this protocol should be directed to the technical contact, Yujia Jiang (jiangyujia@genomics.cn).

Materials availability

All the materials used in this protocol are commercially available.

Stereo-seq: www.stomics.tech/sap/.

Data and code availability

Jiang et al.¹ includes all figures and data generated or analyzed during this study.