GSM7952604: AB5847; Mus musculus; RNA-Seq
Mice were sacrificed at various time points by cervical dislocation. Tumor, spleen, and tdLN were collected and put through a process of mechanical and enzymatic digestion. For single-cell suspensions, tumors were chopped into small pieces and suspended in RPMI supplemented with DNase (2mg/ml, Sigma-Aldrich) and collagenase IV (100mg/ml, Worthington). Tumors were homogenized by GentleMacs tissue homogenizer (Miltenyi Biotec) and incubated at 37oC for 30min, with frequent agitation. Following dissociation procedures, cells were washed with cold PBS, filtered through a 70-μm cell strainer, and centrifuged at 300g for 5 min at 4 °C. Spleen and tdLN were mashed through a 100uM cell strainer and washed with ice-cold MACS buffer (PBS supplemented with 0.2mM EDTA, pH 8, and 0.5% BSA). Cells were centrifuged at 300g, 5 min, and 4°C. Spleen was suspended in red blood lysis buffer (Sigma-Aldrich) and DNase (0.33U/ml, Sigma-Aldrich), incubated for 5min at room temperature, washed with cold PBS, passed through a 70μm mesh strainer, centrifuged at 300g, 5 min, 4°C and then resuspended in ice-cold MACS buffer. Single-cell RNA-seq libraries were prepared as previously described {Jaitin, 2014}. In brief, mRNA from single cells sorted into capture plates were barcoded and converted into cDNA and then pooled using an automated pipeline. 3' end mRNA libraries were prepared for sequencing using MARS-seq (Jaitin et al, Science 2014)