Mucosal cell isolation for scRNA-seq
Mucosal cell isolation for scRNA-seq

Mucosal cell isolation for scRNA-seq

Overview

This protocol walks through the steps involved in preparing cell suspensions for intestinal epithelial cells (IEC), lamina propria (LP), and mesenteric lymph nodes (MLN) collected from naive and infected mice for Cellular Indexing of Transcriptomes and Epitomes (CITE-seq). This protocol was developed, and and serves as the working SOP for single cell work carried out, as part of the Mucosal Immunology Study Team (MIST).

General info

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For successful single cell genomics studies, it is critical that tissues be processed rapidly after dissection from the animal and be kept on cold at all times.
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We have found that the protocol below is best managed by three dedicated personnel. One individual can focus on dead cell removal, while a second individual does cell counting and viability measurements, and the third handles buffers, washes, and centrifugations.
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Mice (6wk old) are ordered from the same room at Jax, which is confirmed to be free of Segmented Filamentous Bacteria (SFB). Upon arrival, mice are acclimated to the animal room for 1-2 weeks before infection. We process two mice in parallel, collecting three tissues from each mouse. We take both mice through the entire protocol below, but then we select the best mouse (based on cell yield and viability) to move forward with processing for 10x CITE-seq. Samples from the unused mouse are discarded.
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Depending on the type of infection, other samples or tissues (e.g. feces) may be collected to confirm infection status in secondary assays carried out at a later time.

Supplies

Reagents

  • Buffer A
    • 1L HBSS + 5% FBS (50mL) + 10mM HEPES (10mL of 1M)
  • Buffer B
    • 1L HBSS + 2mM EDTA (4mL of 0.5M EDTA) + 10mM HEPES (10mL of 1M)
  • Buffer C (for dissociating epithelial cells)
    • 1L HBSS + 5% FBS (50mL) + 5mM EDTA (10mL of 0.5M EDTA)
    • Add DTT right before use (200uL of 15.4mg/mL aliquots which are kept at -20C, final is 0.154 mg/mL)
  • cRPMI (complete RPMI)
    • RPMI+10% FBS + 0.1% β-mercaptoethanol + 1% non-essential amino acids + 1% sodium pyruvate + 1% penn/strep
  • Digestion Buffer (for intestines)
    • 5mL cRPMI + 0.5mg/mL DNase (83uL of 33mg/mL stocks) + 0.167 mg/mL Liberase TL (50uL of 16.67 mg/mL Liberase stock solution)
    • Use 700µL for MLN

Reagent preparation

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DTT has a very short half-life in buffers, so do not add DTT to Buffer C until the day of your experiment
  1. Prepare 10mL of ice cold Buffer A per gut sample.
  2. Prepare 20mL of Buffer C and add 200µL of DTT. Place in 37C water bath.
  3. Reconstitute liberase by adding 300uL cRPMI to a 10mg tube. Can keep at -20 if you don’t use the whole thing.
  4. Make Digestion Buffer fresh by adding Liberase and DNase to cRPMI and place into 37C water bath.

Preparing MLNs

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Be sure to record the weight of each mouse after euthanasia
  1. Remove ileal draining MLN and put into an 1.5mL tube of b on ice.
  2. Move MLN into a prepared tube of 700µL Digestion Buffer. Mince with scissors in this tube of digest buffer and then place at 37C for 20min to digest.
  3. After digestion, pour/pipet the MLN over a 70µM filter over a 50mL conical tube and grind using the back of a 1mL syringe plunger. Rinse the Eppendorf tube out with another 700µL cRPMI and use that to rinse the 70µM filter.
  4. Quick spin the filtered samples without caps with the 70µm strainer still on (get the centrifuge to 600-700 rpm then hit “stop”). This helps pass the sample thru the strainer. Then remove the strainer and spin at 1200 rpm for 5 min at 4C.
  5. Resuspend MLN in 1mL cRPMI and count (live/dead cells) using Trypan blue to determine yield and viability.

Preparation of the small intestine

  1. Cut out whole small intestine from mouse. Fold into thirds and take the final third as the ileum. Remove connective tissue to your best ability (mesenteric fat). Remove Peyer’s patches with scissors and discard.
  2. Open intestine longitudinally with scissors to expose the lumen.
  3. Transfer to ice cold PBS in a Petri dish and manually agitate the dish by hand to loosen and food, feces, or mucous from the lumen. Transfer tissue to a second Petri dish with fresh ice-cold PBS and rinse again.
  4. Cut tissue into quarters and place pieces into 10 mL Buffer A (1 per mouse) on ice.
  5. Repeat the above steps with all intestinal samples
  6. Transfer intestine pieces to pre-warmed Buffer C (1 per mouse) and place on warm shaker at 37C for 20 min.
  7. After 20min, briefly shake with hands vigorously and immediately place samples on ice. Pour off supernatant into the previous 50mL conical tube of Buffer A on ice for each sample.
  8. Add 10mL ice-cold Buffer B to each gut piece. Shake vigorously by hand for 1 min.
  9. Pour off supernatant into the tube of Buffer A where you poured the Buffer C.
  10. Repeat step 8.

IEC isolation

  1. Spin down sample at 1500 rpm for 5 min at 4C. Resuspend in 5mL of Buffer A and filter thru a 70µm strainer into a new 50mL conical tube. Wash the empty tube in 5mL of Buffer A and pass over the strainer.
  2. Quick spin the filtered samples without caps with the 70µm strainer still on (get the centrifuge to 600-700 rpm then hit “stop”). This helps pass the sample thru the strainer. Then remove the strainer and spin at 1500 rpm for 5 min at 4C.
  3. Resuspend in 5mL of Buffer A and filter thru a 40µm strainer into a new 50mL conical tube. Wash the empty tube in 5mL of Buffer A and pass over the strainer.
  4. Quick spin the filtered samples without caps with the 70µm strainer still on (get the centrifuge to 600-700 rpm then hit “stop”). This helps pass the sample thru the strainer. Then remove the strainer and spin at 1500 rpm for 5 min at 4C.
  5. Resuspend samples in 1mL of Buffer A and count with Trypan Blue staining. Count live+dead cells, then proceed to Dead Cell Removal section below.

LP isolation

  1. Add 700µL of Digest Buffer to a 1.5mL eppendorf tube.
  2. After removing IEL/IEC layer, rinse tissue in cold Buffer A in a Petri dish to remove EDTA. Place tissue in 1.5mL tube prepped in Step 1.
  3. Mince tissue manually with scissors in eppendorf tube. Then transfer to the larger 50mL conical tube with the rest of the Digest Buffer (One 50mL conical per sample). Rinse eppendorf tube with 700µL Digest Buffer from this 50mL conical and put back into the conical tube.
  4. Place conical tubes with minced tissue at 37C shaking for 30min.
  5. After digesting, pour tissue over a 70µM filter in a fresh 50mL conical tube and mash tissues through the 70µM filter using a 1mL syringe plunger. Then, rinse with 5mL cRPMI.
  6. Quick spin the filtered samples without caps with the 70µm strainer still on (get the centrifuge to 600-700 rpm then hit “stop”). This helps pass the sample thru the strainer. Then remove the strainer and spin at 1500 rpm for 5 min at 4C.
  7. Resuspend in 2mL of Buffer A and filter thru a 40µm strainer into a new 50mL conical tube. Wash the empty tube in 2mL of cRPMI and pass over the strainer.
  8. Quick spin the filtered samples without caps with the 70µm strainer still on (get the centrifuge to 600-700 rpm then hit “stop”). This helps pass the sample thru the strainer. Then remove the strainer and spin at 1500 rpm for 5 min at 4C.
  9. Resuspend samples in 500µL cRPMI and count with Trypan Blue staining. Count live+dead cells, then proceed to Dead Cell Removal step (see below).

Dead Cell Removal

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Cell counts you get will probably be approximate. If you get >1.5x107 total cells (live+dead), split the cells between 2 columns or otherwise the columns might clog and run slowly
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If necessary, during dead cell removal steps use slow spins (2x at 200 x g for 5min, then 1 x at 100 x g for 5min) to maximize removal of dead cells and debris. More likely to need to do slow spins with the IEC fraction rather than the LP fraction.
  1. Use Miltenyi Dead Cell Removal Kit (see Supplies section above) with MACS LS columns per manufacturers instructions.
  2. After Dead Cell Removal, spin down sample at 1500 rpm for 5 min at 4C, then resuspend sample in 1 mL cRPMI and transfer to an eppendorf tube. Count with Trypan Blue staining.