ATAC-seq Analysis of IFNy Signaling

Cells were pre-treated with JQ1 (1 µM) or A-241 (250 nM) for 1 h prior to the addition of recombinant murine IFNy (1 ng/mL), or vehicle control, for an additional 2 h (3 h total incubation with small molecules). Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq) was performed using an improved protocol to reduce mitochondria from the transposition reaction [40 (link)]]. Briefly, 5e5 MM1.S cells were cultured in duplicate with JQ1, A-485, or DMSO vehicle as described above. Cells were washed once in ice-cold PBS and lysed in ATAC lysis buffer (0.1% Tween-20, 0.1% NP-40, 3 mM MgCl₂, 10 mM NaCl, 10 mM Tris HCl pH 7.4). Tagmentation was then performed with Tn5 transposase and 2 × TD Buffer (Nextera DNA Library Prep Kit, Illumina) for 30 min at 37 °C (in a thermocycler). Tagmented DNA was immediately purified using a MinElute column (Qiagen, #28004) and then amplified for 12 cycles using 2 × KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602) and Illumina-compatible/barcoded primers. The amplified libraries were purified using MinElute columns (Qiagen) and sequenced on an Illumina NextSeq 500 with 75 b.p. single-end reads. Library QC and quantification were performed using D1000 high-sensitivity screen tape with 4200 TapeStation Instrument (Agilent Technologies), and the size was selected between 200 and 500 bp using a Pippin Prep system (Sage Science).