Tn5 transposes

We performed ATAC sequencing on WM989-A6 melanoma cells according to^{22 (link)}. Briefly, we lysed the cells and set up the transposition reaction with the Tn5 Transposes (Illumina Catalog #FC121-1030) at 37℃ for 30 minutes. We cleaned the reaction with a Qiagen MinElute Kit and then amplified the libraries using the custom Nextera PCR primers described in^{22 (link)}. We sequenced our libraries on a NextSeq with 75 base pair reads at a depth of approximately 40–70 million reads per sample. We aligned our reads to hg19 with bowtie2 and then used the HOMER package for peak calling, differential peak calling, motif analysis, and gene ontology analysis (code available at https://bitbucket.org/arjunrajlaboratory/rajlabseqtools). Peaks called as lost or gained if we could identify the peak in one of the conditions and saw a change in read count in the peak of 4 fold or higher across both replicates.

Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was performed on MOLM-13 human AML cells as described(Buenrostro et al., 2013 (link)).Briefly, MOLM-13 cells were subjected to cell lysis in cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Following 30 seconds of lysis, cells were centrifuged at 500 ×g for 10 minutes, 4°C. Lysed cell pellets were then subjected to the transposition reaction at 37°C for 30 minutes using Tn5 Transposes (Illumina Cat #FC-121–1030), followed by purification of transposed DNA using the Qiagen MinElute Kit. Transposed DNA fragment were amplified using NEBNext High-Fidelity 2x PCR Master Mix (New England Labs Cat #M0541) and Customized Nextera PCR primers under the following conditions: (Step 1) 72°C for 5 minutes, (Step 2) 98°C for 30 seconds, (Step 3) 98°C for 30 seconds, (Step 4) 63°C for 30 seconds, (Step 5) 72°C for 1 minute, and (Step 6) 4°C hold, with amplified library purified using Qiagen PCR Cleanup Kit prior to Illumina Sequencing. For PCR steps 3–5, transposed library was amplified with number of cycles based on quantitative PCR in order to stop amplification prior to saturation, thus reducing GC and size bias in PCR.