title: GSM2907067: Cov_Flag_ctnnb1; Mus musculus; ChIP-Seq
library_construction_protocol: RNA-Seq: RNA was isolated by first homogenizing cells in 1ml TRIzol reagent (Invitrogen 15596026) through triturating the cell lysate through a 27-gauge needle a minimum of 7 times. Homogenized samples were mixed with 200µ l chloroform and vigorously shaken by hand for 30sec. Samples were then incubated at RT for 10min before centrifuging at 12,000g for 10min at RT. The aqueous phase containing RNA was transferred to a new tube, mixed with 100µ l chloroform, and subjected to a second round of shaking, incubation and centrifuging.  The aqueous phase was transferred to a new tube containing 1.5µ l 20mg/ml glycogen, and mixed with 450µ l isopropanol to pre- cipitate RNA. Samples were gently mixed and incubated at RT for 10min before centrifuging at 4 °C for 15min. The supernatant was discarded and the pellet was washed by 1ml 75% ethanol. After the wash the RNA pellet was dried at RT for 20min and resuspended in RNase-free water. Yield and purity of RNA was determined by spectrophotometry with Nano drop (Thermo Scien- tific). The RNA was treated with DNase (Promega M6101) ChIP-Seq: 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,