GSM6509788: MC38_Gpr65; Mus musculus; RNA-Seq
At day 17 (B16), respectively at day 16 (MC38) single cell suspensions were prepared from excised whole tumors by tissue digestion using murine tumor dissociation kit and gentleMACS dissociator (Miltenyi Biotec). CD45+ cells were enriched by murine CD45 (TIL) MicroBeads (Miltenyi Biotec) using MACS technology. To enrich purity viable CD45+ cells were sorted using AriaII flow cytometer (BD Bioscience). Capturing of single cells was performed using the BD Rhapsody system (BD Biosciences). For multiplexing samples, tumorinfiltrating CD45+ cells from WT and Gpr65-KO mice were labeled by sample tags against CD45 conjugated with unique oligos (B16_Gpr65 experiment: WT1=Sample tag (ST) 5; WT2=ST6; WT3=ST7, WT4=ST8; KO1=ST9; KO2=ST10; KO3=ST11; KO4=ST12); MC_38_Gpr65 experiment: WT1=ST7; WT3=ST9; KO1=ST10; KO2=ST11; KO3=ST12; Sequences of sample tags can be found under Doc ID 210970) and 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 purifyingWTA 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 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 a HiSeq2500 (B16_Gpr65) using a HiSeq PE Rapid Cluster Kit v2 (PE-402-4002) and combining three HiSeq Rapid SBS Kits v2 (50 cycles). MC38_Gpr65 libraries were sequenced on Illumina's NovaSeq 6000 at Novogene (Cambridge, UK). For processing of the sequencing raw data BD targeted multiplex Rhapsody analysis pipeline version 1.5 was used. For visualization the software Partek Flow (Partek Inc.) has been used.