GSM7047166: mds 6 N12; Homo sapiens; RNA-Seq
Frozen BM cells stored in liquid nitrogen were retrieved right before sample processing. To maximize the cellular viability recovery, samples were processed in batches according to thawing protocol. Briefly, cells were gently thawed in water bath at 37 °C for 5 minutes until partially thawed and immediately placed on ice. Next, cells were gently transferred to 10 ml room temperature warm media (10 ml RPMI1640 supplemented with 20% FBS) and centrifuged (453 g for 5 min). After finishing centrifugation, supernatant was disposed, the cell pellet was carefully re-suspended in 10 ml thawing media (RPMI1640, supplemented with 20% FBS, containing 500 μl Heparin (stock 2 mg/ml, Cat# 9041-08-1; Sigma Aldrich), 20 μl DNase (stock 1 ug/ml, Cat# 89835, Thermo Fischer Scientific) and 200 μl MgSO4 (stock 200 mM, Sigma Aldrich) and incubate in 37 °C for 15 min to digest the DNA released from the dead cells. Following incubation, cells were centrifuged and gently washed twice in 2 ml of 0.04% BSA in PBS. Finally, to remove cell clumps, cells were filtered with 35μm mesh strainer directly to falcon tubes (BD Falcon® 5 ml Round-Bottom Tubes with Cell Strainer Cap, Corning, NY, USA). After transferring cells, 10 ul of cell suspension was stained with 10 ul of 0.4% Trypan blue, counted and assessed for viability using standard hemocytometer and light microscopy. To adjust the cell density, cells were eventually centrifuged again and re-suspended in desired amount of PBS with 0.04% BSA. scRNA-seq libraries were generated using a 10x Genomics Chromium Controller instrument and Chromium Single-Cell 5′ V2 reagent kits (10x Genomics). In brief, cells were concentrated to 1,000 cells per μl and cells were loaded into each channel to generate single-cell Gel Bead-In-Emulsions (GEMs), resulting in mRNA barcoding of an expected 5,000 single cells for each sample. After the reverse transcription step, GEMs were broken, and single-strand cDNA was cleaned up with DynaBeads. The amplified barcoded cDNA was fragmented, A-tailed, ligated with adaptors and amplified by index PCR. cDNA and constructed libraries were assessed by High Sensitivity D5000 DNA Screen Tape analysis (Agilent Technologies) and Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). Sequencing was conducted on an Illumina NovaSeq sequencer with 2 × 100 bp paired reads to reach a depth of at least 50,000 read pairs per cell. The Chromium Single-Cell V(D)J Enrichment Kit was used to enrich immune repertoire, TCR or B-cell immunoglobulin (Ig) transcripts. The single-cell V(D)J enriched libraries were sequenced at 2 × 150 bp with a minimum depth of 5,000 read pairs per cell.