Novaseq s4 300 cycle platform

For single cell RNAseq (scRNAseq) and single cell TCRseq (scTCRseq), viable lymph node FoxP3 GFP⁺ cells were sorted from injured or uninjured Foxp3^DTR mice at 7 days after injury. The sorted FoxP3 GFP⁺ cells were washed and resuspended in sorting buffer at a cell concentration of 1000 cells/µL. About 17,000 mouse cells were loaded onto a 10x Genomics Chromium™ instrument (10x Genomics) according to the manufacturer’s recommendations. The scRNAseq libraries were processed using Chromium™ single cell 5’ library & gel bead kit (10x Genomics). Matched scTCRseq libraries were prepared using 2 µL of post cDNA amplification material and Chromium Single Cell V(D)J Enrichment Kit, Mouse T Cell. Quality controls for amplified cDNA libraries and final sequencing libraries were performed using Bioanalyzer High Sensitivity DNA Kit (Agilent). The sequencing libraries for scRNAseq and scTCRseq were normalized to 4nM concentration and pooled using a volume ratio of 4:1. The pooled sequencing libraries were sequenced on Illumina NovaSeq S4 300 cycle platform. The sequencing parameters were: Read 1 of 150bp, Read 2 of 150bp and Index 1 of 8bp. The sequencing data were demultiplexed and aligned to GRCm38 using cell ranger version 3.1.0 pipeline (10x Genomics).

NUGC3 cells were treated with the IC50 concentration of Cisplatin, Oxaliplatin, 5-FU and DMSO for 2 days. The cells were washed with PBS, trypsinized and labeled with cell hashing antibodies, TotalSeq^TM-C0251 Hashtag 1 (BioLegend #394661) and TotalSeq^TM-C0252 Hashtag 2 (BioLegend #394662). Viable cells were washed and resuspended in PBS with 0.04% BSA at a cell concentration of 1000 cells/µL. About 17,000 viable mouse cells were loaded onto a 10× Genomics Chromium^TM instrument (10× Genomics) according to the manufacturer’s recommendations. The scRNAseq libraries were processed using Chromium^TM single cell 5’ library & gel bead kit (10× Genomics). Matched cell hashing libraries were prepared using single cell 5’ feature barcode library kit. Quality controls for amplified cDNA libraries, cell hashing libraries, and final sequencing libraries were performed using Bioanalyzer High Sensitivity DNA Kit (Agilent). The sequencing libraries for scRNAseq and scTCRseq were normalized to 4 nM concentration and pooled using a volume ratio of 4:1. The pooled sequencing libraries were sequenced on Illumina NovaSeq S4 300 cycle platform. The sequencing parameters were: Read 1 of 150 bp, Read 2 of 150 bp and Index 1 of 8 bp. The sequencing data were demultiplexed and aligned to mm10-3.0.0 using cell ranger version 3.1.0 pipeline (10× Genomics).