Dissociation of adult rodent DRG for single-cell RNAseq

Dissociation of adult mice DRG for scRNAseq

Reagents and animals

1. Mice or rat 8–12-week-old
2. Papain (Worthington Biochemical LS003126 https://www.worthington-biochem.com/PAP/cat.html) 
3. L-Cysteine (Sigma-Aldrich C7352)
4. Collagenase (Sigma-Aldrich C6885-100MG https://www.thomassci.com/Chemicals/Reagent-C/_/SIGMA-Collagenase-from-Clostridium-histolyticum-release-of-physiologically-active-rat-epididymal-adipocytes-tested-Type-II-05-50-FALGPA-units/mg-solid-125-CDU/mg-solid?q=C915Z94)
5. Neurobasal-A (Thermo Scientific 10888022)
6. Hank's balanced salt solution (HBSS) w/o Ca2+ and Mg2+
7. Hepes buffer
8. 0.5M NaOH
9. Bovine Serum Albumin (BSA)
10. Hoechst 33342 Solution

Procedure

A. Preparation of solutions

1. HBSS+H (HBSS with 10% Hepes) 
2. Papain (Make Fresh): 45unit (35ul) Papain + 50ul of 20mg/ml L-cysteine + .5ul .5M NaOH + 3ml HBSS+H. Put on rocker in 4^oC, heat to 37^oC 30 min before use
3. Collagenase (Make Fresh): Dilute 1 4.5mg/200ul into 3ml HBSS+H

B. DRG dissociation

1. Perfuse mice with 10ml HBSS 
2. Dissect L3-5 DRG into a tube containing HBSS+H on ice
3. Transfer DRGs to a 15ml conical containing Papain solution and incubate for 20 minutes at 37ºC
4. Let DRG set at the bottom of the tube then gently remove the supernatant and add 3ml heated HBSS+H
   • Repeat 3x total
5. Gently remove the supernatant, add 3ml of Collagenase solution, and incubate for 20 minutes at 37ºC
6. Gently remove the supernatant and add 3ml heated HBSS+H
   • Repeat 3x total
   • Remove as much supernatant as possible
   • Add 1ml Neurobasal A media then triturate 30x with P1000 until DRGs are completely dissociated and are in a single-cell suspension
7. Pipette the cell solution through a 100um mesh filter into a 50ml conical tube 
   • Wash the filter with 1ml Neurobasal A media
8. Centrifuge at 200 x g for 4 minutes
9. Remove as much supernatant as possible
   • Resuspend cells in Neurobasal A media

C. Cell sorting

1. Centrifuge at 200 x g for 4 minutes
2. Remove as much supernatant as possible
3. Resuspend cells in HBSS+H + 0.1%BSA + Hoechst dye and keep on ice
4. Sort positive cells in UV light, 100um nozzle, into tubes HBSS+H + 0.1%BSA
5. Count manually to make a cell concentration of 1000cells/ul
6. Continue to scRNAseq protocol