GSM3732354: PH001_COG_DIAGNOSIS; Homo sapiens; RNA-Seq
Frozen BM specimens were rapidly thawed and transferred into 50 mL conical tubes. 20 mL Pre-warmed RPMI 1640 (Corning) supplemented with 5% Fetal Bovine Serum (FBS) (Sigma Aldrich) and 2-Mercaptoethanol (2-ME, 55nM) (Thermo Fisher Scientific) was added drop-wise to tubes. Cell suspensions were subjected to centrifugation at 400 x g for 5 min at 4°C and supernatant was discarded. Pellets were re-suspended in 5mL RPMI 1640 with 5% FBS and 2-ME, and filtered using a 70 μm nylon mesh (Thermo Fisher Scientific) with residual cell clumps discarded. After centrifuging at 400 x g at 4°C for 5 min, the supernatant was discarded, and the cell pellet was re-suspended in PBS (Corning) with 2% FCS and placed on ice prior to staining for FACS. Single-cell BM suspensions were re-suspended in PBS with 2% FCS and Human TruStain FcX Blocking Solution (BioLegend) and incubated on ice for 10 mins. Cell suspensions were centrifuged at 400 x g at 4°C for 5 min, and supernatant discarded. Cell pellets were re-suspended in PBS with 2% FCS, FITC-conjugated anti-human CD45 (1:200, BD Pharmingen), APC-conjugated anti-human CD19 (1:200 concentration, BioLegend) and PE-conjugated anti-human CD10 (1:200, BioLegend), and incubated on ice for 30 mins. Following incubation, cell suspensions were subjected to two wash cycles involving re-suspension of cell pellets in 3 mL PBS with 2% FCS, followed by centrifuging at 400 x g at 4°C for 5 min, followed by supernatant removal. Finally, samples were re-suspended in 300 mL of PBS with 2% FCS per 2 x 10e6 cells, supplemented with DAPI (0.5 mg/mL) for live/dead staining. Cell sorting was performed using the SY3200™ highly automated parallel sorting (HAPS) cell sorter (Sony). Single Cell 3' Reagent Kits v2: Chromium™ Single Cell 3' Library & Gel Bead Kit v2, PN-120237; Single Cell 3' Chip Kit v2 PN-120236 and i7 Multiplex Kit PN-120262" (10x Genomics).
GEO Accession
GSM3732354