GSM8676382: Knockout, Day8, biol rep 1; Mus musculus; RNA-Seq
Bulk RNA sequencing: Total RNA was isolated using the RNeasy Micro kit (Qiagen) according to manufacturer's instructions. Single cell RNA+V(D)J sequencing: For scRNAseq, tumors were digested in DMEM+++ supplemented with Liberase TL (Merck, 10mg/mL) and DNAse I (Roche, 20mg/mL) for 30min at 37°C 5% CO2. 0.5M EDTA was added for 5min before meshing tumors through a 20µm cell strainer (Corning). Tumor single cell suspensions were stained using CD45 PE (Biolegend, 1:200) for 20min at 4°C. CD45+ leukocytes were flow sorted. Adequate Cell viability and concentration was verified with trypan blue staining and TC10 automated cell counting (Biorad) complemented with hemocytometer visual inspection. Bulk RNA sequencing: Libraries were prepared using the QuantSeq 3′ mRNA-Seq Library Prep +UMI method (Lexogen GmbH, Vienna Biocenter). Quality-validated, barcoded libraries were multiplexed and sequenced using Illumina NovaSeq technology. Single cell RNA+V(D)J sequencing: Cell suspensions were processed with a Chromium single cell controller (10xGenomics) and Chromium Next GEM Single Cell 5' Kit v2 chemistry, targeting 4000-6000 cells per sample. The resulting total RNA and V(D)J libraries were multiplexed and sequenced with Illumina NovaSeq technology, generating in total approximately 700 M read pairs.