GSM7874169: HL, case 1, scRNAseq; Homo sapiens; RNA-Seq
Each tissue sample was placed in a sterile Petri dish on ice. The tissue was subsequently minced to smaller pieces of less than 3 mm. Sample pieces were transferred to a gentle MACS C Tube (Miltenyi #130-093-237) containing 5 ml digestion enzyme mix. The C tube was then placed on a gentleMACS Octo Dissociator for mechanical dissociation. Upon run completion, the sample was incubated for 30 min at 37°C under continuous low shaking. Subsequently, the sample was filtered using a 70-mm nylon mesh (Miltenyi #130-095-823). 10 ml of this cell suspension was counted by Trypan Blue to determine the concentration of live cells. The sample was then centrifuged at 300 x 3 g and 4°C for 5 min, and the supernatant was discarded. The cell pellet was resuspended in 1 ml freezing media (Gibco). Single-cell RNA-seq libraries were prepared with Chromium Single cell 30 Reagent v2 Kits according to the manufacturer's protocol. The Chromium Single Cell 30 Library and Gel Bead Kit v2 (PN-120237), Chromium Single Cell 30 Chip kit v2 (PN-120236) and Chromium i7 Multiplex Kit (PN-120262) were used. Briefly, single-cell suspensions were loaded on the Chromium Single Cell Controller Instrument (10 × Genomics) to generate single cell gel beads in emulsions (GEMs). After generation of GEMs, reverse transcription reactions were engaged barcoded fulllength cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads Myone Silane Beads (Thermo Fisher Scientific). cDNA was then amplified by PCR with appropriate cycles which depend on the recovery cells. Subsequently, the amplified cDNA was fragmented, end-repaired, A-tailed, index adaptor ligated and library amplification.