GSM7029187: Reactive LN stromal cells processed fresh_1; Homo sapiens; RNA-Seq
Fresh or thawed cryopreserved samples were incubated in 37°C water bath with 3mL enzymatic digestion solution. Enzymatic digestion of fresh or thawed, cryopreserved tissue was performed otherwise similarly to prior descriptions (31, 32). An enzymatic medium was prepared by adding 2 mg/mL dispase II (Gibco), 0.6 mg/ml collagenase P (Sigma Aldrich), and 0.3 mg/mL DNase I (Sigma Aldrich) to pre-warmed RPMI with 2% FBS and 20mM HEPES. Every 10 minutes, samples were agitated by pipetting using a 1000µl micropipette. Every 15 minutes, the digestion medium with cells in suspension was aspirated, transferred through 70µm filter to 20mL ice-cold FACS buffer (PBS containing 2% FBS, 20mM HEPES, and 2mM EDTA) before being spun at 1500rpm for 5 minutes and resuspended in fresh FACS buffer. Fresh digestion medium was immediately added to the residual tissue for a total of three serial, 15-minute digestions.  Flow cytometric cell sorting was performed with the FACSDIVA software on a BD FACSAria (Franklin Lakes, NJ) set to 20 psi with a 100µm nozzle. The sorting protocol was as previously described, although we did not require any bead-based enrichment prior to sorting and could compensate successfully with antibody-stained UltraComp eBeads Compensation Beads (Invitrogen, Waltham, MA) (31). We sorted ≥ 30,000 CD45- EpCAM- live singlet cells into 1.5mL DNA LoBind tubes (Eppendorf, Hamburg, Germany) containing 40µl FBS. Purity was routinely > 95%. Tubes were spin-concentrated with only a part of the supernatant removed by gentle aspiration to leave ~50µl volume in the tube. The concentrated cells were then fully resuspended in the remaining volume and counted with a hemacytometer using a Trypan Blue viability dye. Flow cytometric analysis was performed using FlowJo v.10.6.1 (Ashland, OR). Library preparation was performed according to the manufacter's instructions (single cell 3' v3.2 protocol, 10x Genomics).