GSM4726951: SUB07865: Control_vs_AsiC_treatment_CD45pso_cell_comparision; Mus musculus; RNA-Seq
On day 14, tumors were harvested, incubated at 37°C for 15 min with 100 mg/ml Liberase TL (Roche) diluted in Ca2+ and Mg2+-free RPMI (ThermoFisher), followed by shaking at 37°C for 10 min. Samples were then filtered twice through 40 mM strainers. Dead cells were removed and CD45+ cells were enriched with CD45 microbeads at 4°C. More than 95% of cells were CD45+ as verified by flow cytometry. The enriched cells were diluted to 200,000 cells/ml in Ca2+ and Mg2+-free RPMI medium containing 1% FBS and kept on ice until the cells were flowed into the microfluidic device. 6,000 cells per sample were encapsulated using the inDrop technology (Klein et al., Cell, 2015) Single-cell transcriptomes were barcoded within the microfluidic droplets. After within droplet reverse transcription, emulsions of approximately 3,000 cells were broken and used for library preparation as described (Klein et al., Cell, 2015; Zilionis et al., Nature Protocols, 2017),. Libraries were indexed with V3 sequencing adaptors, pooled from different samples at equimolar ratios, and sequenced on an Illumina NextSeq 500 system using the NextSeq 75 High Output Kits and standard Illumina sequencing primers and 61 cycles for read 1 and 14 cycles for read 2, 8 cycles each for index read 1 and index read 2.