Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Chromatin Immunoprecipitation was performed using the following protocol. 4 x 107 formaldehyde-fixed cells were lysed in SDS lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, supplemented with Complete, EDTA free protease inhibitor cocktail (Roche)) and diluted 4-fold with dilution buffer (16.4 mM Tris-HCl, pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, supplemented with inhibitors). The lysate was sonicated in aliquots using a water bath sonicator (Diagenode Bioruptor Pico) for 11 cycles (30s on/30s off) and cleared by centrifugation. The combined sonicated material was further diluted 1.5-fold in dilution buffer and 1.7 x 107 cell equivalents were subjected to immunoprecipitation with 5 μg (α-H3K27Ac, α-H3.3, α-RPB1, α-S5P CTD and α-S2P CTD), 7 μg (α-Spt5) or 1:50 dilution (α-H3K36me3) of antibodies at 4°C overnight. The next day, immune complexes were incubated with 30 µl magnetic beads (Protein G Dynabeads, Thermo Fisher Scientific) or (Protein A/G magnetic beads, Pierce). The magnetic beads were washed twice with low salt wash buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), twice with ChIP wash buffer (50 mM HEPS, pH 7.6, 500 mM LiCl, 1 mM EDTA, 0.7% sodium deoxycholate and 1 % Igepal CA-630) and twice with TE buffer with 50 mM NaCl. Chromatin Immunoprecipitation was performed with the SimpleChIP Enzymatic Chromatin IP Kit (#9003) according to the manufacturer's protocol with minor modifications. In brief, 40 x 10e6 Ramos cells were spun down at 90xg for 10 min before the pellet was reconstituted in 9 ml of RPMI with 2% FBS in a 15 ml conical tube. Then, 0.6 ml of 16% Formaldehyde (Pierce/ThermoFisher #28906) was added and the cells were placed on a rocker for 10 min at RT. One ml of 10x glycine solution was added to quench the reaction, and the cells were then returned to the rocker for 5 min at RT before being spun down at 300xg and washed 2x with PBS. All following steps were performed on ice or at 4°C unless otherwise noted. Cells were reconstituted in 10 ml of Buffer A with protease inhibitors and allowed to rest for 10 minutes and the lysed cells were spun down at 2000xg for 5 min to pellet nuclei. Pelleted nuclei were washed with 10 ml of Buffer B, pelleted at 2000xg for 5 min, and then resuspended in 1 ml of Buffer B. 1.7 µl of micrococcal nuclease (CST #10011) was added to the nuclei and the tube incubated in a 37°C water bath for 20 min. The reaction was stopped with 100 µl of 0.5 ml of EDTA, and the nuclei pelleted at 16,000xg for 1 min. The pellet was then resuspended in 1 ml of 1x ChIP Buffer with protease inhibitors and sonicated in 200 µl aliquots in a Qsonica sonicator for 2 cycles of 15 s on, 45 s off at 20% power. The lysate was then clarified at 10,000xg for 10 min before the supernatant was removed and diluted five-fold in 1x ChIP Buffer with protease inhibitors. For each ChIP, 500 µl of chromatin was incubated with 1-2 µg of antibody in a 1.5 ml Eppendorf tube at 4°C overnight on a rotator. The next day, 30 µl of Protein G Magnetic beads (CST #70024) was added to each tube and the mixture incubated at 4°C for 2 hours on a rotator. The beads were pelleted using a magnetic separation rack and the supernatant discarded. The beads were then washed 3x with a low-salt wash and 1x with a high-salt wash using 5 min incubations on a rotator. Chromatin was eluted from beads with the addition of 150 µl of 1x Elution Buffer and incubation at 1200 rpm on a thermal mixer for 2 hours at 65°C. The beads were pelleted and the supernatant was moved to a new 1.5 ml Eppendorf tube and 2 µl of Proteinase K (CST #10012) added. The mixture was incubated for 2 hr at 65°C and then DNA was purified using SimpleChIP DNA Purification Buffers and Columns (CST #14209) and eluted in a volume of 50 µl. ChIP-seq libraries were prepared for each ChIP from three independent ChIP experiments using TruSeq ChIP Sample Preparation kit (Illumina) or NEBNext Ultra II DNA Library Prep kit (New England Biolabs) according to manufacturer's instructions.