title: s_48_Marcel_Abb52_LND1-3_GEX
design: 8- to 10-week-old male WT C57BL/6J mice were implanted s.c. with 1 million TRAMP-C2 or MC38 cells and treated i.p. with Isotype or CPI (anti-CTLA4+antiPD-1) on days 3, 6 and 9, followed by vehicle (DPBS) or 20 mg/kg ABT-317 on day 12. On day 15 after implantation, tumor,  and TDLNs from mice were harvested and processed to single cell suspension as described. To enrich for CD45+ cells in tumors, total tumor lysates were stained for Live/Dead and CD45. Live CD45- or CD45+ (tumors)  were sorted with a FACS Aria into separate tubes and barcoded by with mouse TotalSeq-C03 hashtag antibodies 1-10 (155861, 155863, 155865, 155867, 155869, 155871, 155873, 155875, 155877, 155879, Biolegend) at a 1:200 dilution for 40 minutes at 4C to identify individual samples and treatment groups. Then, individual reactions of 6 x 104 cells each with 1:1 CD45+:CD45- (tumors) ratios (unsorted total cells for TDLNs) and equal hashtag representation for batch correction purposes were prepared at the Chromium Controller (10X Genomics) using the Chromium Next GEM Single Cell 5' Kit v2 (1000263, 10X Genomics) and the Chromium Single Cell Mouse TCR Amplification kit (1000254, 10X Genomics) and sequencing was performed on an Illumina NovaSeq 6000 with paired-end 100 base pair read lengths.