GSM2087619: Dox_2_ChIP-Seq; Mus musculus; ChIP-Seq
Cells were cross-linked with 1% formaldehyde for 10 minutes with rocking at room temperature, and cross-linking was terminated by addition of 125 mM glycine. Pellets were washed in cold PBS and snap frozen in liquid nitrogen. Cell pellets were lysed in buffer (50 mM Tris-HCl [pH 8.1], 10 mM EDTA, 1% SDS) containing Halt™ protease inhibitor mixture (Thermo Scientific). Chromatin fragmentation was conducted using 30 cycles of sonication on a Bioruptor (Diagenode). PU.1-chromatin complexes were immunoprecipitated overnight a 4°C with Rabbit polyclonal anti-mouse PU.1 (T-21) (Santa Cruz Biotechnology) or mouse control IgG (Abcam) conjugated to protein G dynabeads (Invitrogen, Burlington) on 300 ug of chromatin per sample. Bound beads were washed once in low salt wash buffer (0.1% SDS, 1% Triton-X-100, 2 mM EDTA, 20 mM Tris-HCl[pH 8.0], 150 mM NaCl), once with high salt wash buffer (0.1% SDS, 1% Triton-X-100, 2 mM EDTA, 20 mM Tris-HCL[pH 8.0], 500 mM NaCl), once with LiCl buffer (0.25 M LiCl, 1% NP-40, 1% Na-deoxycholate, 1mM EDTA, 10 mM Tris-HCl [pH 8.0]), and twice with Tris-EDTA buffer at pH 8.0. Immunocomplexes were eluted in elution buffer (1% SDS, 0.1 M NaHCO3) and the buffer adjusted to 200 mM NaCl. Cross-linking was reversed at 65°C overnight and DNA isolated using a Wizard SV Gel and PCR purification kit (Promega). Libraries were generated robotically with 10 ng of fragmented DNA (100-300 bp) using the Kapa HTP Library Preparation Kit (Kapa Biosystems) as per the manufacturer’s recommendations except that adapters and PCR primers were diluted 100-fold, the size selection step was done after the PCR step and the number of PCR cycles increased by 6. Adapters and PCR primers were purchased from Integrated DNA Technologies whereas size selection has been performed on a Pippin Prep instrument (SAGE Biosciences Inc). Libraries were quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies) and the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (D-Mark). Average size fragment was determined using a LaChip GX (PerkinElmer) instrument. Libraries were sequenced on a single-end 100bp read length run on a HiSeq2000 (Illumina).
GEO Accession
GSM2087619