GSM7679699: mixed sample of MC38mOVA_Control_DIVA2treated_day16; Mus musculus; RNA-Seq
At day 16, respectively day 20, single cell suspensions were prepared from excised whole tumors by tissue digestion using Collagenase 4 (800 U/ml) and DNAse I (1:100) and gentleMACS dissociator (Miltenyi Biotec). CD45+ cells were enriched by murine CD45 (TIL) MicroBeads (Miltenyi Biotec) using MACS technology. Capturing of single cells was performed using the BD Rhapsody system (BD Biosciences). For multiplexing samples, tumorinfiltrating CD45+ cells from WT mice were labeled on d16 or d20 by sample tags against CD45 conjugated with unique oligos as indicated in the processed data file section. Sequence of sample tags can be found under BD; Doc ID 210970. Cells were loaded on BD Rhapsody cartridge. Capture of single RNA molecules on barcoded magnetic Oligo-dT- beads and cDNA synthesis was performed as described in the manual (Single cell capture and cDNA synthesis with the BD Rhapsody single-cell analysis system; Doc ID: 210966 Rev. 1.0). mRNA and Sample Tag Libraries were prepared following the manual (mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library Preparation Protocol; 23--21712-00). Upon purifying WTA and Sample tag library, concentration of libraries was assessed by Qubit 2.0 using the DNA high sensitivity kit and average library size was determined on a bioanalyzer 2100 using a high-sensitivity DNA chip. Final sequencing library was prepared by dilution and pooling of mRNA and Sample tag library following the recommendations of the sequencing preparation sheet provided by BD. The libraries were sequenced on Illumina's NovaSeq 6000 at Novogene (Cambridge, UK). For processing of the sequencing raw data BD Rhapsody WTA analysis pipeline version 1.9 was used. Single-cell RNA-sequencing data were processed with R (further described below) and visualized using the R package iSEE.