GSM6704896: Fresh_Lin_Depleted_scRNA-seq; Mus musculus; RNA-Seq
For scRNA-seq extract protocol, For C57BL/6 Lineage-depleted BM cells were seeded at 1E3 cells per 35 mm dish in StemCell Technologies' MethocultTM GF M3434 methylcellulose media and cultured for two weeks with either 30 uM SB204990 (Tocris Cat# 4962) or an equivalent volume of DMSO. Cells were harvested and resuspended in PBS alongside freshly isolated lineage-depleted BM cells from a C57BL/6 mouse. Cells were then stained for viability using eBioscience™ Fixable Viability Dye eFluor™ 780 (Thermo Fisher Scientific 65-0865-14) and flow-sorted for viable cells. For scATAC-seq extract protocol, C57BL/6 Lineage-depleted BM cells were seeded at 1E3 cells per 35 mm dish in StemCell Technologies' MethocultTM GF M3434 methylcellulose media and cultured for two weeks with either 30 uM SB204990 (Tocris 4962) or an equivalent volume of DMSO. Cells were harvested and resuspended in PBS alongside freshly isolated lineage-depleted BM cells from a C57BL/6 mouse. For scRNA-seq library construction, Cells were submitted to Vanderbilt University's VANTAGE Core, where they were prepared for single-cell 5' RNA sequencing using the 10x Genomics Chromium system. Libraries were prepared using P/N 1000014, 1000020, 1000080, and 120262 according to the manufacturer's protocol. The libraries were sequenced using the NovaSeq 6000 with 150 bp paired-end reads. RTA (version 2.4.11; Illumina) was used for base calling. For scATAC-seq library construction, Nuclei were isolated and submitted to Vanderbilt University's VANTAGE Core, where they were prepared for scATAC-seq following the manufacturer's protocol using P/N 1000111, 1000086, and 1000084.