title: GSM5788454: H3K27ac-DMSO (Control for Bortezomib); Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP assays were carried out according to an optimized version of the protocol provided with the iDeal ChIP-seq Kit for Transcription Factors (Diagenode). Following proteasome inhibition for 3 h with 25 µM Lactacystin, 60 nM Bortezomib, 60 nM Carfilzomib or 0.1% v/v DMSO control, MM.1S, MM.1S-Siah2, MM.1S-shHDAC3, and control cells were collected in 50 mL conical tubes, counted, and assessed for cell viability. For each immunoprecipitation, 25 million cells were harvested, washed once in sterile 1X PBS (Phosphate-Buffered Saline), resuspended in 1% formaldehyde in PBS solution, and fixed for 10 min. at room temperature with gentle end-over-end rotation on a Hula mixer (10 rpm). Cross-linking reaction was quenched by adding glycine at a final concentration of 125 mM to the fixation solution for additional mixing (5 min, 10 rpm). After incubation, fixed samples were washed once in sterile 1X ice-cold PBS and stored on ice until further processing. Cell lysis was carried out with ice-cold lysis buffers iL1b and iL2, according to vendor's protocol for suspension cells. After centrifugation, nuclear pellets were resuspended in SDS-containing shearing buffer iS1b supplemented with protease inhibitor cocktail at a concentration of 1.5 million cells per 100 µl buffer iS1b. Nuclear cell suspension was split into 250 µl aliquots and chromatin was sheared using a Bioruptor Pico water bath sonicator (Diagenode). To ensure generation of 150-300 bp DNA fragments suitable for next generation sequencing, the Bioruptor Pico sonicator was set at 10 cycles, each cycle 30 s “ON” and 30 s “OFF”, and kept at 4°C. After sonication, samples were centrifuged (10 min, 16,000 g, 4°C) to remove nuclear membrane debris and insoluble fraction. Supernatant (sonicated chromatin) was stored at -80°C into “Input” aliquots or used immediately in immunoprecipitation (IP) reactions, which were carried out with 2 µg of antibodies/IP and DiaMag Protein A-coated magnetic beads (30 µl/IP) under constant rotation on a Hula mixer (10 rpm). The ChIP-grade primary antibodies used in this study were the following: anti-H3K4me1 antibody (Abcam, ab8895), anti-H3K4me3 antibody (Abcam, ab8580), anti-H3K27ac antibody (Diagenode, C15410196), and anti-HDAC3 antibody (Millipore Sigma, 17-10238). After immunoprecipitation overnight at 4°C, the immunoprecipitated complexed were captured with a magnetic rack, washed, and eluted according to Diagenode's protocol. ChIP and “Input” samples were then de-crosslinked overnight at 65°C in a temperature-controlled water bath. The next day, ChIP and “Input” DNA was recovered and purified using iPure beads provided in the manufacturer's kit. Finally, a 55%-25% DNA fragment double size selection was performed using Agencourt AMPure XP beads (Beckman Coulter) and final amounts of size-selected DNA were measured on a Qubit fluorometer (Thermo Fisher Scientific). The purified and size-selected DNA was then included in library preparation for next generation sequencing. Single-indexed DNA libraries were constructed with the Ultra Next DNA library prep kit I and II (New England Biolabs (NEB), E7370S and E7645S) and prepared for multiplex sequencing using NEBNext Multiplex oligos (E7335S and E7500S) following manufacturer's instructions. Library quality control, including assessment of fragment size distribution and quantification by qPCR, and library sequencing was conducted by the Baylor College of Medicine Genomic and RNA Profiling Core. The size distribution of the final libraries were confirmed by electrophoretic analysis on an Agilent Bioanalyzer 2100 prior to sequencing. Briefly, 1.8 pM of equimolarly pooled libraries with 1% PhiX control spike-in were loaded onto a NextSeq 500 high output v2.5 flow cell (Illumina, 20024906) and analyzed on a Illumina NextSeq 500 sequencing system. The flow cell was sequenced in a 75 bp single-end run, enabling the generation of a minimum of 25 million reads per sample. DNA libraries prepared for sequencing on a Illumina HiSeq 2500 were processed identically for quality control. After equimolar pooling of the individual samples, a 10 pM library with 5% PhiX control spike-in was sequenced on a Illumina HiSeq 2500 instrument as a 1 x 50 bp single-end sequencing run (25 million reads per sample) in rapid run mode (v.2). Clustering and sequencing performance were controlled as previously described (Sweeney M.A. et al., Sci. Rep, 2020, PMID 32811853). Libraries were sequenced on the Genome Analyzer following Illumina's protocols for ChIP-seq with HiSeq 2500 or NextSeq 500.