Illumina NovaSeq 6000 paired end sequencing; Single cell GET-seq analysis of cancer cell lines upon administration of FOLFOX or FOLFIRI at different time points
Single cell GET-seq analysis of 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 permeabilized nuclei following the “Nuclei Isolation for Single Cell ATAC Sequencing” protocol CG000169, Rev D (10X Genomics). Specifically, we prepared the nuclei suspension in order to get 5'000 nuclei as target nuclei recovery. Single-cell GET-seq was performed on Chromium platform (10x Genomics) following “Chromium Single Cell ATAC Reagent Kits” user manual version CG000168 Rev C. Tn5 and TnH were used in substitution of the 10X ATAC enzyme (10X Genomics) in the transposition mix assembly step. When a sequential Tn5 to TnH reaction was performed, a transposition mix containing 1.5 uL of Tn5 was incubated 30 min at 37 °C, then 1.5 uL of TnH was added and the reaction was continued for a total of 1 h incubation.