AB1 and renca tumours were surgically removed 1 h prior to ICB administration and immediately submerged in cold PBS, cut into 1–2 mm pieces with a scalpel blade and dissociated using the GentleMACS system (Miltenyi). Cell suspensions were frozen in RPMI medium containing 50% FCS and 10% DMSO. Cryopreserved single cell suspensions were rapidly thawed in a 37 °C water bath and prepared for single cell library construction as previously described73 (link). Libraries were constructed using the 10X Chromium 3’ workflow (version 2 chemistry) as per the manufacturer’s directions. We aimed to capture 9000 cells per sample. Libraries were quantified using the TapeStation D1000 kit (Agilent). Sequencing was performed by Novogene, using NovaSeq S2 flowcell sequencing protocols.
For single cell analysis, we processed FASTQ files from 6 AB1 and 6 Renca samples using cellranger v3.0 (10X genomics). For each sample, we performed demultiplexing and read alignment using the cellranger count function, using cellranger’s pre-supplied mm10 reference with an expect-cells parameter of 6000.
For single cell analysis, we processed FASTQ files from 6 AB1 and 6 Renca samples using cellranger v3.0 (10X genomics). For each sample, we performed demultiplexing and read alignment using the cellranger count function, using cellranger’s pre-supplied mm10 reference with an expect-cells parameter of 6000.
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