GSM4593540: scRNA-seq_Sample 1; Mus musculus; RNA-Seq
After flow sorting, cells from each sample was resuspended in PBS containing 2% FCS and stained with oligo tagged TotalSeq antibodies directed against CD45 for 30 minutes on ice. Cells were washed and pooled accordingly, centrifuged at 1200rcf for % minutes at 4 degrees and resuspending in 31.7 ul of PBS + 2% FCS. For each 10X channel 6 samples were combined: WT1-Tumour, dLN, ndLN; KO1 Tumour, dLN, ndLN; WT2-Tumour, dLN, ndLN; KO2 Tumour, dLN, ndLN. Single-cell RNA-Seq libraries were generated from each time point using the 10X Genomics Chromium Controller Instrument (10X Genomics, Pleasanton, CA) and ChromiumTM Single Cell 3' Reagent Kits v2  according to manufacturer's instructions. Reverse transcription and sample indexing was performed using the C1000 Touch Thermal cycler with 96-Deep Well Reaction Module. Briefly, the suspended cells were loaded on a Chromium controller Single-Cell Instrument to first generate single-cell Gel Bead-In-Emulsions (GEMs). After breaking the GEMs, the barcoded cDNA was then purified and amplified. The amplified barcoded cDNA was fragmented, A-tailed and ligated with adaptors. Finally, PCR amplification was performed to enable sample indexing and enrichment of the 3' RNA-Seq libraries. The final libraries were quantified using Thermo Fisher Qubit dsDNA HS Assay kit (Q32851) and the fragment size distribution of the libraries were determined using the Agilent 2100 BioAnalyzer High Sensitivity DNA kit (5067-4626). Pooled libraries were then sequenced using Illumina Sequencing By Synthesis (SBS) chemistry. Bulk RNA-seq libraries were generated from QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen). Libraries were made according to manufacturer's protocol and sequenced on illumina NextSeq 550.
GEO
GSM4593540