Illumina NovaSeq 6000 sequencing; Single cell tracking of expressed barcodes in cancer cell lines upon administration of FOLFOX or FOLFIRI at different time points
Single cell tracking of expressed barcodes in cancer cell lines upon administration of FOLFOX or FOLFIRI at different time points
Cells were trypsinized, harvested and washed with PBS/0.04% BSA and immediately used. HCT116 cells were grown in DMEM supplemented with 10% Fetal Bovine Serum. Cells were plated, and after 24h were treated with FOLFOX or FOLFIRI at the following concentration: 5-FU 100µM + OXA 100µM (FOLFOX) or 5-FU 100µM + SN-38 100nM (FOLFIRI) for 8h or 16h. Then, cells were harvested for experimental purposes. For long term (2 weeks) treatments, cells were plated and exposed to 5-FU 10µM + OXA 1µM (FOLFOX) or 5-FU 10µM + SN-38 10nM (FOLFIRI). Nucleic acids were directly processed on intact cells following standard Chromium Single Cell 3' Reagent v3 chemistry protocol (10X Genomics) using  Chromium platform (10x Genomics). Target cell recovery was 5'000. Starting libraries prepared using Chromium Single Cell 3' Reagent v3 chemistry protocol (10X Genomics). cDNA were then amplified with KAPA HiFi HotStart ReadyMix (Roche) by using the following primers:  5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCGACCACCGAACGCAACGCACGCA-3' and ACACTCTTTCCCTACACGACGCTCTTCCGATCT, at the following PCR conditions: 95°C for 3 minutes, 98°C for 20 seconds, 65°C for 15 seconds, 72°C for 45 seconds and back to step 2, 13x followed by a 2 minute hold at 72°C. After a cleanup-SPRI beads selection, the eluted samples were again amplified by using P5 primer 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA-3' and multiplexed with ten different primers indicated in Supplementary Information and obtained from Illumina Inc. using the same PCR conditions and 6x rounds. The product was again purified by cleanup-SPRI beads selection.