GSM8112304: GM08505, BLM-/-, biological replicate 1, IP rep2, G4 ChIPseq rep2; Homo sapiens; ChIP-Seq
Chromatin preparation essentially follows the previously described protocol in {https://doi.org/10.1038/nprot.2017.150} with a few modifications. Cells cultured in two full 15cm dishes were washed once in 1xPBS and directly fixed in the dish with 1% formaldehyde in cell culture medium at RT for 8.5 min. Nuclei were lysed in 250ul lysis buffer and sheared in small tubes (microTUBE AFA Fiber Pre-Slit Snap-cap 6x16mm, Covaris, Cat. 520045) on Covaris S220 with the following parameter: water level=15, duty cycle=15, intensity =6, cycle per burst =200 for 3min. There was a 30s pause after 1 min of sonication. The IP reaction was performed essentially as described previously in {https://doi.org/10.1038/nprot.2017.150}, with the following modifications. For each sample, 2 biological replicates were prepared, each in turn divided into 3 technical replicates of immune-precipitation (IP), except for the wild-type fibroblasts, for which the second biological replicate was performed with only 2 technical replicates due to the limited amount of BG4 antibody. During BG4 IP, chromatin was incubated with the primary antibody for an extended duration of 2 hours, and a doubled amount of beads was used in pull-down. To purify DNA from both the IP samples (DNA pulled down by the beads) and the input samples (sheared chromatin without going through IP steps), 75 μl of reverse-crosslinking buffer (0.2% SDS, 1x TE, and 50 mM NaCl) was introduced. The samples were incubated at 37°C for one hour, followed by an overnight incubation at 65°C. Following an additional hour of proteinase K digestion at 65°C, DNA purification was carried out using a MinElute kit (QIAGEN). Library preparation were carried out with the DNA Thruplex kit (Takara, Cat. #R400674, Cat. #R400665) following the manufacturer's protocols.