GSM1646846: T-Blast CD4+PMA Runx1; Mus musculus; ChIP-Seq
Chromatin was prepared and immunoprecipitation was carried out as previously described (Lichtinger et al) for the antibodies, H3K27ac (ab4729) and H3K4me2 (Millipore 07-030). For all other antibodies, JunB (sc-46), Runx1 (ab23980), Ets1 (sc-111), and BRD4 (Bethyl laboratories), chromatin was prepared as follows. Cells were resuspended in PBS at 3.3x106 cells/ml and cross-linked with 0.83mg/ml disuccinimidyl glutarate (DSG) for 45 minutes at room temperature with rotation. Cells were washed four times with PBS and further crosslinked in PBS at 2x106 cells/ml with 1% formaldehyde for 10 minutes at room temperature. Crosslinking was quenched with 0.125M glycine, cells were washed twice in PBS and incubated with Buffer A at 4oC for 10 minutes followed by buffer B at 4oC for 10 minutes. Double cross-linked chromatin was then used for immunoprecipitation as previously described (Lichtinger et al). Libraries were prepared from approximately 10ng DNA from ChIP samples. Libraries for sequencing on the Solid platform were prepared according to the manufacturer’s instructions. Samples were sequenced using 50bp reads. All other libraries were prepared using the Tru-Seq library preparation kit according to the manufacturer’s instructions (Illumina). These samples were sequenced on the Illumina HiSeq platforms using 50bp single end reads