GSM6560826: Day22, differentiating sample, scRNAseq; Homo sapiens; RNA-Seq
Medium was removed and cells were washed one time with DMEM/F12 and one time with sterile PBS, and incubated in 2 mg/mL Papain solution (STEMCELLTM, Cat. 07465) for 7 minutes at 37 ℃. An equal volume of 1% BSA in PBS without Ca2+ and Mg2+ was added to the cell suspension, and cells were suspended by pipetting up and down approximately 15 times using a 1 mL pipette tip. The suspension was moved to a 2 mL Eppendorf tube, and centrifuged at 300 x g for 3 minutes. The supernatant was removed and cells were resuspended in 1% BSA + 2 µM EDTA in PBS without Ca2+ and Mg2+, where volume was adjusted based on a viable cell count of approximately 1000 cells/µL using LUNA-FLTM Dual Fluorescence Cell Counter. The cell suspension was filtered 2-4 times through a 35 µm cell strainer tube cap (Falcon, Cat. 352235), and processed for scRNA-seq library construction and sequencing. Single-cell RNA-sequencing was performed using the Chromium Single-Cell 3′RNA-sequencing system (10x Genomics, Pleasanton, CA) with the Reagent Kit v3 according to the manufacturer's instructions. Briefly, the cells were loaded into Chromium Single Cell B Chip (10x Genomics, Pleasanton, CA) and gel beads in emulsion (GEM) generation were performed aiming at 10,000 cell capture per sample. Subsequent cDNA purification, amplification, and library construction was performed as instructed. Sample libraries were sequenced on Illumina NovaSeq 6000 system using following read lengths: 28bp (Read 1), 8bp (i7 Index), 0 bp (i5 Index) and 89bp (Read 2) aiming at 50 000 reads per cell sequencing depth.