GSM4146637: ChIP-HELIOS; Homo sapiens; ChIP-Seq
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. libraries were prepared from 50 ng of eluted DNA using the Ultra II DNA Library Prep Kit for Illumina (NEB #E7645) following the manufacturer's protocol
GEO Accession
GSM4146637