title: GSM9429774: Lung, Progressive disease, Pre-treatment, TMRE low; Homo sapiens; RNA-Seq
library_name: GSM9429774
library_construction_protocol: Kidney and lung cancer patient peripheral blood was collected in mononuclear cell preparation tubes (BD Biosciences, 362761) and processed according to manufacturer protocol. Healthy donor PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (Cytiva,17144002) in SepMate-50 tubes (STEMCELL Technologies, 85450) following manufacturer protocols. For single cell experiments, kidney and lung patient PBMCs were FACS sorted using BD FACS Aria II at the Vanderbilt Flow Cytometry Shared Resource by cell singlets (FSC-A vs FSC-H and SSC-A vs SSC-H), lymphocytes, viable, CD3+, TMRElow and TMREhigh. TMRElow and hi populations were separated based on TMRE modal distributions. Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 5' v2 platform (P/N: 1000263; 10x Genomics, Inc.) according to the manufacturer's protocol.  FACS-sorted CD3+ TMRE low and CD3+ TMRE high T cells from patient PBMCs were used as input for scRNA-seq library preparation were loaded into each channel of the Chromium X controller to achieve the target cell recover of ~5,000 cells.  Cells were partitioned into Gel Bead Emulsion (GEMs), where cell lysis and barcoded reverse transcription of the RNA occurred, followed by cDNA amplification.  Amplified cDNA was purified using SPRI beads and dual indexed libraries were constructed by fragmentation, end repair, A-tailing, adapter ligation, and PCR amplification. The library quality was assessed using a Bioanalyzer and quantified using a qPCR-based method with the KAPA Library Quantification Kit (P/N: KK4873) and the QuantStudio 12K instrument. Prepared libraries were pooled in equimolar ratios, and the resulting pool was subjected to cluster generation using the NovaSeq 6000 System, following the manufacturer's protocols. 150 bp paired-end sequencing was performed on the NovaSeq 6000 platform targeting 50,000 reads per cell.  Raw sequencing data (FASTQ files) obtained from the NovaSeq 6000 was subjected to quality control analysis, including read quality assessment. Real Time Analysis Software (RTA) and NovaSeq Control Software (NCS) (1.8.0; Illumina) were used for base calling. MultiQC (v1.7; Illumina) was used for data quality assessments.