Dissociation of Adult DRG for scRNAseq and Cultures

Reagents and animals

Mice or rat 8–12-week-old

Media for dissociation

• Neurobasal A media (Thermo Scientific 10888022)
• HBSS+H: HBSS (w/o Ca²⁺ and Mg²⁺;  and 10% Hepes)

Papain (Make Fresh)

45unit Papain (35ul) (Worthington Biochemical LS003126) + 50ul of 20mg/ml L-cysteine (Sigma-Aldrich C7352) + .5ul .5M NaOH + 3ml HBSS+H

• Put on rocker in 4C, heat in incubator 30 min before use

Collagenase (Make Fresh)

Dilute 1 4.5mg/200ul (Sigma-Aldrich C6885) into 3ml HBSS+H

Media and plates for cultures

Total Volume = 3ml + (volume/well)*(# wells)

• Neurobasal A– Total calculated volume
• Pen/Strep 1:100
• B27 Plus (Thermo A3582801) 1:50
• Glutamax (Thermo 35-050-061)1:100
• FBS 5%

Plates

1. Coat plates for minimum 4 hours at 37ºC with Poly-D-Lysine (PDL; Thermo ICN10269490) 1mg/ml diluted 1:10 in sterile water.
2. Wash 3x with water for 3 minutes each (can be stored at -20 ºC)

Procedure

Dissociation

1. Perfuse animals with cold HBSS
2. Dissect L3-5 DRG into a tube containing HBSS+H on ice
3. Transfer DRGs to a tube containing heated Papain solution and incubate for 20 minutes at 37ºC
4. Gently remove the supernatant and wash with 3ml heatedHBSS+H
   1. Repeat 3x total
5. Gently remove the supernatant, add 3ml of heated Collagenase solution, and incubate for 20 minutes at 37ºC
6. Gently remove the supernatant and add 3ml heatedHBSS+H
   • Repeat 3x total
   • Remove as much supernatant as possible
   • Add 1ml Neurobasal A media then triturate 30x with P1000
7. Pipette the cell solution through a 100um mesh filter into a 50ml conical
   Wash the filter with 1ml Neurobasal A media

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