GSM9046461: Mouse HSPC, RNA, Day 5; Mus musculus; RNA-Seq
Cells were collected, filtered (40 micron), pelleted and resuspended in staining buffer (PBS + 2% BSA + 0.02% Tween). Cells were then Fc blocked (10 min @ 4C) and then stained with appropriate hashtag antibody (6 in total for each of 6 genotypes, staining for 30 min at 4C). Cells were then washed 4x, resuspended in 'Submission Buffer' (PBS + 0.04% BSA), and pooled equally to a concentration of 3,000 cells/microliter prior to submission. Single-cell encapsulation and scRNA-seq library prep of was performed on the Chromium instrument (10x Genomics) following the user manual (GEM-X Single Cell 3' Reagent Kits v4). Each sample loaded onto the cartridge contained approximately 30,000 cells (hashed samples) at a final dilution of ~3000 cells/μl. Transcriptomes of encapsulated cells were barcoded during reverse transcription and the resulting cDNA was purified with DynaBeads, followed by amplification per the user manual. Next, the PCR-amplified product was fragmented, A-tailed, purified with 1.2X SPRI beads, ligated to sequencing adapters and indexed by PCR. Indexed DNA libraries were doublesize purified (0.6–0.8X) with SPRI beads and sequenced on an Illumina sequencer (R1 – 26 cycles, i7 – 8 cycles, R2 – 70 cycles or higher) to a depth of >50 million reads per sample (>13,000 reads per cell) at MSK's Integrated Genomics Operation Core Facility.