GSM5331340: VCaP ERG ChIP; Homo sapiens; ChIP-Seq
Nuclear lysates were incubated with 5uL of indicated antibody and 50uL of Dynabeads for 4 hours in dilution buffer(20mM Tris, 2mM EDTA,150mM NaCl, 1% Triton X-100,  2 mg/mL BSA) followed by 4 washes with wash buffer(20mM Tris, .25% NP-40, .05% SDS,  2mM EDTA, 250mM NaCl). ChIPs were then incubated with RNAse A (5Prime) and Proteinase K (Sigma) followed by DNA extraction with Qiagen PCR purification kit. Total Cellular RNA was extracted using Qiagen RNeasy kit followed DNAse treatment (Turbo) and  poly-A selection using oligo dT beadsd (Ambion). Poly-A selected RNA was then reverse transcribed using random hexamer primers and SuperScript III. Double stranded DNA was then produced from cDNA fragments using DNA ligase and DNA polymerase. Isolated DNA was sonicated for 30 minutes (30sec on / 30 sec off) followed by End Repair with T4 DNA polymerase, Klenow DNA polymerase, and T4 PNK. End repaired DNA was 3' Adenylated with Klenow exo (3' to 5') followed by ligation of Illumina TruSeq adaptors with T4 DNA ligase. Ligated DNA fragments were subject to 15 cycles of PCR with Q5 polymerase followed by size selection with AMPure XP beads. ATAC-Seq libraries were generated using ActiveMotif ATAC-Seq kit. 50,000 cells were spun down, washed with PBS, and lysed with ATAC-Lysis buffer followed by tagmentation. Tagmented DNA was purified and PCR amplified for 10 cycles followed by SPRI bead cleanup.