GSM9109287: AB15410.txt; Mus musculus; RNA-Seq
Tumors were minced into small pieces on ice and digested using RPMI supplemented with 1mg/ml Collagenase IV (LS004188, Enco) and 0.05mg/ml DnaseI (DnaseI, Sigma Aldrich) at 37 degrees for three times 8 min. In between the incubation steps, the tissue was further dissociated using a gentleMACS dissociator (130-093-235, Miltenyi Biotec). After digestion, the cells were filtered trough a 100um cell strainer and washed with ice cold PBS.  Cells were stained with FACS antibodies and viability dye for 30min on ice. After washing them again with ice cold PBS, cells were sorted and acquired on a BD FACS Symphony 6 flow cytometer. After excluding doublets and dead cells, two 384-well plates of CD45+ cells and CD90.2+ and/or NK1.1+ (=T/NKT/NK/ILCs) were sorted per sample The 384-well plates contained a lysis solution, mineral oil and barcoded poly(T) reverse transciption primers enabeling scRNA sequencing. SPID-seq92, an adapted version of the MARS-seq pipeline93, was used to generate scRNA-seq libraries. In short, polyadenylated mRNA from individual cells that had been sorted into 384-well plates was captured, followed by a barcoding step of the mRNA during reverse transcription into cDNA. cDNA of each plate was pooled into a single tube, and was fragmented and amplified to generate sequencing-ready libraries for NovaSeq X plus (Illumina) sequencing. Quality control was performed on each library created from a plate.