GSM5068767: IgG ChIP - HepG2 cells; Homo sapiens; ChIP-Seq
Briefly,  cells from four 100 cm Petri dishes at 70-80% confluence were crosslinked in 1% formaldehyde for 10 min with continuous shaking. Crosslinking was stopped by addition of 0.15 M glycine while continuing shaking. After collecting cells by scraping, pellets were washed 3x with cold PBS. Nuclei were isolated and lysed and the obtained chromatin was sonicated in fragments of approximately 200 bp using the Bioraptor instrument. Number of cycles and settings for sonication were previously described(53).  At the same time, antibodies used for immunoprecipitation were coupled with magnetic protein G beads (Invitrogen 100-03D) by incubating 75 μl of protein G beads with 10 μg of anti-HOXA13 antibody (anti human HOXA13 abcam, #ab106503), anti-HA tag antibody (abcam anti-HA tag ChIP grade  #ab9110)  or 10 μg of mouse IgG (Santa Cruz Biotechnology, #sc-2025) as a negative control for 1h at room temperature with constant rotation. At the end of the sonication process, an aliquot of chromatin was kept as input control for each sample and an equal amount of sonicated chromatin was incubated with magnetic beads-antibody previously coupled at 4°C overnight while rotating. After incubation, samples were washed several times and then eluted according to the protocol described by Blecher-Gonen et al.(52).  All the samples including the input were processed for RNase and Proteinase K treatment, followed by overnight reverse cross-linking at 65°C with continuous shaking. DNA purification followed using Agencourt AMPure XP (A63880 Beckman Coulter). Library construction was performed using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® preparation kit according to the standard protocol. Massively parallel sequencing was performed on an Illumina NextSeq 550 according to manufacturer's instructions. 50 million reads per sample were obtained.