title: GSM4809403: ES_2i_control_H3K4me3_1; Mus musculus; ChIP-Seq
library_construction_protocol: A total of 2×10^7 ES cells were used per ChIP assay. Cells were washed with 1 x PBS and crosslinked with 1% paraformaldehyde for 10 min at RT and quenched with 1ml 2.5M glycine. Cells were washed with cold PBS three times and resuspended in 300μl lysis buffer. The cell suspension was sonicated in Bioruptor Pico sonication device with the setting of 30 sec on, 30 sec off for 16 cycles followed with centrifuga- tion at maximum speed for 10 min. The supernatant was saved and mixed with antibody coated protein G dynabeads(Invitrogen 10003D) at a ratio of 5μl per 1μg antibody for immunoprecip- itation overnight at 4°C . The next day the protein G dynabeads were washed with 800μl RIPA buffer three times. After the elution, 4μ l RNase A(10μ g/ml) was then added with the beads being incubated at 37°C for 2 hours, after which 2μl Protease K(Invitrogen 20mg/ml) was added and the beads were incubated at 55 °C for 2 hours. After the RNase A and Protease K treatment, the beads were incubated at 55 °C overnight for decrosslinking. The next day the DNA on beads was extracted by 300 μl phenol chloroform isoamyl alcohol(25:24:1) with centrifugation at 12,000 rpm for 5 min at RT. The supernatant was transferred to a new 1.5ml tube with 12μl of 5M NaCl and 2μl glycogen (20mg/ml). The DNA was precipitated by 750μl cold 100% ethanol with at least 30 min incubation at - 80 °C . The sample was centrifuged for 30 min in 4 °C at maximum speed and the ethanol was decanted. The pellet was washed with 800 μ l cold 70% ethanol, centrifuged and air dried. The DNA was resuspended in 55μl nuclease-free water. RNA-seq and ChIP-seq libraries were prepared using the TruSeq RNA kit for sequencing,