GSM8513136: OSRC2, HIF2a-KI, Rep1; Homo sapiens; ChIP-Seq
For ChIP sample preparation, 1×107 adherent cells were washed with PBS, trypsinized, transferred to Eppendorf tubes and collected by centrifugation at 300×g for 5 min at RT. Cell pellets were washed once with RT PBS-complete [PBS supplemented with 1× protease inhibitor (Sigma PIC0006) and 5mM sodium butyrate], resuspended in 2 mM of disuccinimidyl glutarate (DSG, Thermo Fisher Scientific 20593) diluted in PBS, and then agitated at 850 rpm on a Fisherbrand Isotemp Heat/Cool Programmable Thermal Mixer II (Thermo Fisher Scientific) at RT for 45 min. The cell pellets were collected by centrifugation at 300×g for 5 min at RT. The supernatant was removed, and the cell pellets were resuspended in freshly made 1% formaldehyde (Thermo Fisher Scientific 28908) diluted in PBS followed by agitation at 850 rpm on a Fisherbrand Isotemp Heat/Cool Programmable Thermal Mixer II for 10 min at RT. The crosslinking was quenched by adding glycine to a final concentration of 0.125 M with agitation at 850 rpm on a Fisherbrand Isotemp Heat/Cool Programmable Thermal Mixer II for 5 min at RT. The cell pellets were collected by centrifugation at 300×g for 5 min at 4°C. Cell pellets were washed once with ice-cold PBS-complete. Crosslinked cells were then pelleted, snapfrozen and stored at -80°C.   Aliquots of frozen pellets were thawed on ice, resuspended in 500 l of 1% SDS lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS, 1× protease inhibitor, 5 mM sodium butyrate) and lysed on ice for 10-15 min. The lysates were transferred to 1 ml Adaptive Forced Acoustics (AFA) fiber milliTUBEs (Covaris 520135) and sonicated at the following settings: 140 peak incident power, 5% duty factor and 200 cycles per burst) using Covaris E220 sonicator. Sonication lasted for 10 min. Post sonication, the lysates were transferred to Eppendorf tubes and cleared by centrifugation at 17,000×g for 15 min at 4°C.   To determine the amount of precleared chromatin to be used for ChIP-Seq experiments, 10 l of the lysates were aliquoted (INPUT) and 90 l of TE was added to make 100 l solution. 1 l RNase A (Invitrogen AM2696) was added to the samples and incubated for 30 min at 37°C, then 5 l Proteinase K (20 mg/ml, Thermo Fisher Scientific EO0492) was directly added to the mixture and incubated at 65°C for 16 h with constant agitation at 850 rpm on a Fisherbrand Isotemp Heat/cool Programmable Thermal Mixer II at the indicated temperatures. Next the chromatin was purified using PCR Purification Kit (Qiagen 28104). Briefly, 500 l PB buffer was added to each 100 l sample and the mixture was mixed and loaded onto the purification column. The purified chromatin was eluted using 32 l EB buffer. Chromatin (INPUT) concentration was determined using a Qubit High Sensitivity Kit (Life Technologies A33231) on a Qubit 4 Fluorometer (Invitrogen Q33239). 40 g chromatin (INPUT) was immunoprecipitated with 5 g of FLAG antibody (Sigma-Aldrich F1804). Antibody-bead conjugation was done by first taking 30 l of protein G beads (Life Technologies 10004D) per sample and washing them three times with 500 l ice-cold 0.5% BSA diluted in PBS. The beads were then resuspended in 500 l ice-cold 0.5% BSA diluted in PBS with the desired number of antibodies and rotated end-to-end for 4-6 h at 4°C, followed by two additional washed with ice-cold 0.5% BSA diluted in PBS to remove unbound antibodies. Lysates containing the desired amount of chromatin/protein were diluted 10-fold using ChIP dilution buffer (20 mM Tris-HCl pH 8.0, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 1× protease inhibitor, 5 mM sodium butyrate), added to pre-washed antibody-bead conjugates and then rotated end-to-end overnight at 4°C. The next day, the bead-bound chromatin-antibody complexes were washed twice with RIPA 0 buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA pH 8.0, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), twice with RIPA 0.3 buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA pH 8.0, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 0.3 M NaCl), twice with LiCl buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA pH 8.0, 0.5% NP40, 0.5% sodium deoxycholate, 0.25 M LiCl), and then eluted with 100 l pre-warmed (37°C) ChIP elution buffer (1% SDS, 0.1 M NaHCO3 in H2O). 1 l RNase A was added to the samples and incubated for 30 min at 37°C, then 5 l Proteinase K was added to the samples and incubated at 65°C for 16 h. For RNase A and Proteinase K incubation, the samples were constantly agitated at 850 rpm on Fisherbrand Isotemp Heat/cool Programmable Thermal Mixer II. The supernatant was purified using a MinElute PCR Purification Kit (Qiagen 28004), eluted in 12 l H2O, and quantified using a Qubit High Sensitivity Kit (Invitrogen Q33230). A 5 l sample was used for ChIP-seq library preparation with a Swift DNA Library Prep Kit (Swift Biosciences 1002). 75 bp paired-end reads were sequenced on a NextSeq instrument (Illumina).