GSM1573759: D3.75 Bry+ cells 48h+Dox ChIPSeq; Mus musculus; ChIP-Seq
For ChIPSeq, 10 million cells were trypsinized and fixed with 11% formaldehyde in media for 20 minutes with rotation. The fixation reaction was stopped by adding 125 mM Glycine solution and rotating the mixture for 5 minutes at room temperature. Cells were then quick frozen and stored at -80ºC. Cells were thawed and lysed first in 200 μl of lysis Buffer I, followed by 200 μl of lysis buffer II. The resulting nuclear pellets were suspended in 135 μl of lysis buffer III. Sonication was performed using the Covaris sonication instrument using the following settings: Time: 10 minutes, Duty cycle: 5%, Intensity: 4, Cycles per burst: 200, Temperature: 4ºC. Finally, 65 μl of the sonicated solution was used to perform the ChIP pull down using Protein G Dynabeads conjugated to anti-Flag antibody, overnight at 4ºC. Cells without Dox were used as negative control. For ChIPSeq, DNA library preparation was done using the NEB ChIP-Seq sample preparation kit. The libraries were run on HiSeq 2000 machines at the genomics shared resource facility (SRF) at Icahn School of Medicine at Mount Sinai, NY. The concentration of the libraries was assayed using Agilent Bioanalyzer and sequenced using 1*100bp on a HiSeq 2000 platform
GEO
GSM1573759