GSM2691366: CD34_pos_ATACseq; Mus musculus; ATAC-seq
A total of 107 cells were used per ChIP experiment. Cells were cross-linked in 1% formaldehyde for 10 min at RT with rotation followed by addition of 0.125 M glycine to quench the cross-linking reaction. Cells were pelleted and washed twice with ice-cold PBS. Nuclei were isolated using nuclei lysis buffer and chromatin DNA was then sheared using a Bioruptor® sonicator (Diagenode). A sample of 1/100 volume of the sheared DNA was reserved for use as the input control sample. ChIP antibodies (SOX4, STAT3, and GABPα) or normal rabbit IgG were added to Dynabeads Protein A (Invitrogen) beads and incubated for 4 h at 4 °C with rotation. The Dynabeads-antibody complexes were then incubated with the sheared chromatin DNA overnight at 4 °C. After immunoprecipitation, RNase A and Proteinase K were added to the precipitated complex and the samples were incubated at 65 °C overnight with shaking to reverse the crosslinks. For ATAC-Seq, 50,000 cells separated by FACS were spun down and washed with cold PBS and then 50 μl of cold lysis buffer was added to the cell pellet. The Nextera DNA Library Prep Kit (Illumina) was used for the transposition reaction. The transposed DNA was purified using a Qiagen PCR Purification Kit (Qiagen). Purified DNA fragments were then amplified using NEBNext High-Fidelity PCR Master Mix (New England Biolabs). qPCR was performed to determine the appropriate number of additional PCR cycles, allowing us to stop amplification prior to saturation. A total of 10^5 cells were used for ATAC-Seq experiment. Cells were separated using MACS and FACS. The ChIP-Seq libraries were prepared using the DNA SMART ChIP-Seq Kit (Clontech Laboratories) according to the manufacturer's instructions.  ATAC-Seq library was prepared following a published protocol (PMID:25559105). Libraries were quantified using the KAPA Library Quant Kit for Illumina Sequencing Platforms (KAPA BIosystems). All sequencing libraries were sequenced on an Illumina HiSeq 2000 Sequencer.