GSM8514907: ICI-related pneumonitis patient 2, TCR-seq; Homo sapiens; RNA-Seq
A total of 30ml of lavage fluid was retrieved from each patient, with 5-10ml reserved for routine clinical tests and the remainder used for scRNA-seq. The collected BALF samples were encapsulated using 20ml centrifuge tubes and immediately placed on ice. The cooled BALF samples were mixed with RPMI 1640 complete medium and filtered through a 100mm nylon cell strainer to obtain single-cell suspensions. To minimize the potential for blood cell contamination, red blood cells were removed using Red Blood Cell Lysis Buffer. After two washes with 1x PBS (Invitrogen), the cell pellets were resuspended in sorting buffer (PBS supplemented with 1% FBS). Based on fluorescence-activated cell sorter (FACS) analysis, single CD45+cells from each patient's samples were sorted using anti-human CD45 antibodies (Biolegend) into 1.5 ml low binding tubes (Eppendorf) with 50 ml of sorting buffer for scRNA-seq. Cell suspensions were barcoded through the 10x Chromium Single Cell platform using Chromium Next GEM Single Cell 5' Kit v2. Single cells were washed once with PBS containing 0.04% bovine serum albumin (BSA) and resuspended in PBS containing 0.04% BSA to a final concentration of 500-1200 cells/ml as determined by Rigel S2 cell counter (Countstar). The loaded cell numbers ranged from 300-500000 aiming for 300-14000 single cells per reaction. 5000 cells were captured in droplets to generate nanoliter-scale Gel bead in EMulsion (GEMs). GEMs were then reverse transcribed in a C1000 Touch Thermal Cycler (BioRad) programmed at 53℃ for 45 min, 85℃ for 5 min, and held at 4℃. After reverse transcription and cell barcoding, emulsions were broken and cDNA was isolated and purified with Cleanup Mix containing DynaBeads and SPRIselect reagent (ThermoFisher Scientific), followed by PCR amplification. Amplified cDNA was then used for 5' gene expression library construction. For RNA-seq library construction, amplified cDNA was fragmented and end-repaired, double-sided size-selected, PCR-amplified with sample indexing primers, and repeated double-sided size-selection. For T-cell receptor (TCR) library construction, TCR transcripts were enriched from amplified cDNA by PCR. Subsequently, enriched PCR product was fragmented and end-repaired, size-selected, PCR-amplified with sample-indexing primers, and size-selected. Libraries prepared according to the manufacturer's instructions were then purified and profiled for quality assessment. Single-cell RNA and TCR V(D)J libraries were sequenced by an Illumina NovaSeq 6000 sequencer with 150 bp paired-end reads.