GSM8927084: 4T1 TILs, PEG+ADU, Whole Transcriptome; Mus musculus; RNA-Seq
Fresh 4T1 tumors treated with PEG+PBS or PEG+ADU-S100 were pooled (n=4 per group) and mechanically and enzymatically disaggregated in dissociation buffer consisting of DMEM full media + 100 U/mL collagenase type IV and 50 μg/mL DNase I. Suspension cells were incubated at 37°C for 45 minutes and then further mechanically dissociated. Red blood cells were removed before Live/Dead staining (Biolegend, Cat.# 423111) and Fc blocking. Cells were then stained with antibodies specific for mouse CD45 (Biolegend, Cat.# 103106) and mouse CD3 (Biolegend, Cat.# 100219). Samples were washed and sorted using a BD FACS Melody instrument to isolate viable CD45+ and CD3+ cells. Cells were then washed and assessed for viability. 16,500 cells per condition were loaded onto a 10X Chromium instrument (10X Genomics) per the manufacturer's instructions. Single-cell RNA and TCR libraries were generated using the Chromium Single Cell 5′ Library Construction (10X Genomics, Cat.# PN-1000190) and Chromium Single Cell Mouse TCR Amplification kits (10X Genomics, Cat.# PN-1000254) per user guide. Quality control of the completed libraries was performed using the Bioanalyzer High Sensitivity DNA Kit (Agilent, Cat.# 5067–4626) and then sequenced using the Illumina NovaSeq platform.