title: GSM7112140: 20230206_CGCR035_HCC827_K27ac; Homo sapiens; OTHER
library_construction_protocol: ATAC-Seq: The Omni-ATAC protocol (Corces et al., 2017) was used with slight modifications as follows. 100,000 cells per condition were used and washed with PBS before lysis. Cell pellets were lysed in 50μl of cold ATAC-seq resuspension buffer containing 10mM Tris–HCl pH 7.4, 10mM NaCl and 3mM MgCl2, supplemented with fresh NP-40 (final 0.1% v/v), Tween-20 (final 0.1% v/v) and digitonin (final 0.01% v/v) for 3–5min on ice. Next cells were washed with 1ml of resuspension buffer supplemented with Tween-20 (final 0.1% v/v) and pelleted at 500g for 10min at 4 °C. Cell pellets were resuspended in 50μl of transposition reaction mix containing 25μl of 2× TD buffer, 2.5μl of transposase, 16.5μl of 1× PBS, 0.5μl of 1% digitonin (final 0.01% v/v), 0.5μl of 10% Tween-20 (final 0.1% v/v) and 5μl of nuclease-free water. The transposition reaction was incubated at 37 °C for 30min with constant shaking on a thermomixer. The Qiagen MinElute Reaction Cleanup Kit was used for DNA purification. A standard ATAC-seq amplification protocol with 5-7 cycles of amplification was used to amplify the tagmented library (Buenrostro et al., 2015). RNA-Seq: One million cells were harvested from from PC9*, HCC4006 and HCC827* parental and osi- or gefitinib- resistant cell line pairs and washed with cold PBS to remove trypsin and stored in RLT buffer (Qiagen) until further processing. RNA was purified using the Qiagen RNeasy kit, and further processed using the Illumina NEBNext Ultra II Directional RNA Library Prep Kit. CUT&RUN: 500,000 cells per sample were harvested from PC9*, HCC4006 and HCC827* parental and osi- or gefitinib- resistant cell line pairs. CUT&RUN was performed following EpiCypher's protocol within their CUTANA ChIC/CUT&RUN kit with slight modifications to day 1. Prior to wash steps, cells were lysed using a nuclear extraction buffer 20mM HEPES–KOH, pH 7.9, 10mM KCl, 0.1% Triton X-100 and 20% glycerol, supplemented with fresh 0.5mM spermidine, and cOmplete Mini, EDTA-free protease inhibitor. Next, cells were pelleted at 500g for 3 mins. Cell pellets were then washed using protocol's permeabilization buffer and subsequently incubated with activated Concanavalin A beads at room temperature for 15 mins. Next, beads were mixed in Antibody Buffer and incubated with appropriate primary antibodies overnight (antibodies in key resource table). CUT&RUN libraries were prepared with Epicypher's CUTANA ChIC/CUT&RUN Library Prep Kit and sequenced on Illumina Next-seq 500 using 37 bp paired-end sequencing parameters.     ATAC-seq samples were sequenced on Illumina NovaSeq 6000 Next Gen Sequencing using 37 bp paired-end sequencing parameters.    RNA-seq libraries were prepared using NEBnext Ultra II Directional RNA Library Prep Kit for Illumina using standard protocols. RNA was sequenced on Illumina Next-seq 500 using 75 bp single-end sequencing parameters.