scRNAseq of CD45+ cells from Bladder Cancer Patient PBMC and Tumor and Healthy Donor NK Cells
Description
This dataset is associated with the manuscript https://www.biorxiv.org/content/10.1101/2024.04.30.591366v2 Sample preparation for scRNASeq For BCa patients, live, CD45+ singlets were FACS-sorted (BD FACSAria II, 100µM nozzle, 4° C) from either Ficoll-isolated PBMC or freshly dissociated tumor tissue. Cells were sorted into DPBS + .04% BSA, resuspended at 1000 cells/µL, and loaded onto a Chromium controller (10x Genomics) for encapsulation into droplets, lysis, barcoding, reverse transcription, and library preparation using Single Cell 3' v2 reagents. For HD NK cells, PBMC were isolated as above, followed by depletion of non-NK lineages using immuno-magnetic beads (EasySep human NK cell enrichment kit, Stemcell Technologies). Samples were sequenced in dedicated flow cells on a HiSeq 2500 (Illumina) to an average depth of 91,787, 76,918, and 71,692 reads/cell for HD NK cells, BCa patient PBMC, and TIL respectively. Pre-processing and analysis of scRNASeq 10x Genomics 3' sequencing was performed. Raw Illumina sequencing data was converted to FASTQ, demultiplexed and aligned to the GRCh38 reference genome using CellRanger v3.0.0 (10x Genomics).
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Steps to reproduce
Sample preparation for scRNASeq For BCa patients, live, CD45+ singlets were FACS-sorted (BD FACSAria II, 100µM nozzle, 4° C) from either Ficoll-isolated PBMC or freshly dissociated tumor tissue. Cells were sorted into DPBS + .04% BSA, resuspended at 1000 cells/µL, and loaded onto a Chromium controller (10x Genomics) for encapsulation into droplets, lysis, barcoding, reverse transcription, and library preparation using Single Cell 3' v2 reagents. For HD NK cells, PBMC were isolated as above, followed by depletion of non-NK lineages using immuno-magnetic beads (EasySep human NK cell enrichment kit, Stemcell Technologies). Samples were sequenced in dedicated flow cells on a HiSeq 2500 (Illumina) to an average depth of 91,787, 76,918, and 71,692 reads/cell for HD NK cells, BCa patient PBMC, and TIL respectively. Pre-processing and analysis of scRNASeq Raw Illumina sequencing data was converted to FASTQ, demultiplexed and aligned to the GRCh38 reference genome using CellRanger v3.0.0 (10x Genomics)