Day +1, Processing Dispase samples for scRNA-seq
Biopsies incubated in 0.4% Dispase in HBSS overnight at 4C (or 1h at RT)
Collagenase aliquots and Trypsin-EDTA aliquots (see above)
DNase I (Thermo Scientific 90083 – says 2500 U/uL but is actually ~5000 U/uL): -20C
FBS (Atlanta Biologicals S11550): -20C in aliquots
Complete media (e.g. IMDM +10% FBS +P/S, complete KSFM, complete RPMI + GlutaMAX, etc.)
Razor (or scalpel) blades x2
70 nm nylon mesh strainers x2
0.5 mL Eppendorf tube x1 per sample labeled for scRNA-seq
Protocol for cell isolation from skin biopsy for scRNA-seq:
- Warm complete media, FBS, aliquots x2 each of Collagenase and Trypsin-EDTA
- To each 5mL aliquot of Collagenase or Trypsin, add 10U/mL DNase solution and invert to mix
- Calculate quantity based on U on slip (~11uL for 50,000U in 5mL)
- Clean MACSMix and place in 37C incubator
- Pour 70% EtOH into 10 cm plate to wash blades; lean blades on edge of second plate to dry
- Swirl Dispase and pour biopsy into 10 cm plate
- With two pairs of forceps, peel epidermis off of dermis
- Transfer epidermis to Trypsin-EDTA + DNase and rock at highest (top) MACSMix setting at 37C for 1h
- Set timer for 1h to quench Trypsin-EDTA with FBS
- Transfer dermis to new 10 cm plate with drop of Dispase to avoid drying
- Using an ethanol-sterilized razor (dried with Kimwipe) and forceps, mince dermis into 1-2 mm cubes
- Transfer pieces Collagenase + DNase; pipet some out to rinse mincing dish and add back to tube
- Rock at highest MACSMix setting at 37C for 1.5h-2h
- While samples are rocking, submit samples to sequencing core
- When epidermis has incubated 1h, quench with 5 mL warm FBS
- Can process immediately or return to 37C rocker until dermis is ready
- 5min before dermis is done, strain epidermis solution through 70 um mesh into labeled 50 mL conical
- Use back of syringe plunger to shred epidermis against nylon mesh, then rinse mesh with 2 mL IMDM
- Repeat several times; leftover tissue on mesh is okay
- Process dermis in the same way into the same 50mL conical, shredding and rinsing several times
- Spin down samples at 1800 RPM for 6 minutes at 4C (RT okay)
- For each, aspirate liquid and re-suspend in 500 uL (small pellet) to 1mL (larger pellet) of IMDM + 10% FBS
- Pipet to mix, then transfer 12 uL resuspended cells and 12 uL Trypan blue to Eppendorf tube and mix
- Add 10 uL to each side of hemocytometer and count live and dead cells
- Live cells exclude blue dye (clear/shiny); blue cells are dead
- If <25% of cells are dead, omit live/dead column – calculate cells per uL
- Count # cells per 9 center squares = x (20x = # of cells per uL)
- If >25% of cells are dead, consider live/dead column (see below) with caveat that this will increase handling/incubation times and likely increase cell death and bias recovery toward hardier cell types
- Prepare cooler with wet ice
- If combining cells 1:1, calculate the number of uL for 20,000 epidermal cells and 20,000 dermal cells and combine in an Eppendorf tube
- Bring volume up with media such that you have 40 ul (seeking concentration of 1000 cells/ul)
- If sample is too dilute or concentrated, processing time will be increased at the core
- Typically, sequencing core prefers 1000 cells/uL but can accommodate some variability, Err on the side of more concentrated – typically core prefers to avoid spinning the samples
- If sufficient cells, can do 40,000 and 40,000 in 80 uL, as core prefers to have plenty of cells
- If insufficient cells, try to give at least 20,000 in 20 uL and warn core
- Bring an aliquot of spare media for core to dilute samples
- Label samples on lid with sample ID from submission
- Transport samples and spare media to sequencing core on wet ice
- At the core, staff will count cells/check viability while you wait to ensure you want to proceed
- Auto-count will show populations of larger cells (live) and smaller cells (“dead”)
- Note: smaller “dead” cells are most likely lymphocytes in our samples; Trypan blue exclusion is MUCH more reliable – do not be concerned