GSM4217577: 2-HiChIP_1hr_YY1; Mus musculus; OTHER
In situ contact libraries were generated according to the in situ Hi-C published protocol through proximity ligation with modifications. Cells are lysed and chromatin restriction digested using MboI. Biotin labeled dATP is used to fill in the restriction fragment overhangs and mark the DNA ends. Ends are then proximity ligated using DNA ligase. After proximity ligation, nuclei are pelleted and nuclei lysed. Nuclei are then sonicated to solubilize chromatin. Protein A beads are used to clarify supernatant for chromatin immunoprecipation step. Samples are incubated overnight with antibody at 4C with rotation. DNA is eluted off beads and supernatant transferred to new tubes. Crosslinks are reversed. DNA is then purified using Zymo DNA purification kit according to manufacturer's protocol and eluted in water. Streptavidin C-1 beads are used to capture fragments with biotin-dATP labeled ends. After capture, beads are separated with a magnet and the supernatant is discarded. Beads are then washed in several buffers. Beads are resuspended in master mix containing Tn5 Tagment DNA enzyme, which is essential for adapter ligation to samples. Beads are washed several buffers, then separated on a magnet and supernatant discarded. To generate the sequencing library, PCR amplification of the tagmented DNA is performed while the DNA is still bound to the beads. Beads are resuspended in a PCR master mix, consisting of a unique Nextera Ad2.X primer, Phusion HF 5X buffer, dNTPs, universal Nextera Ad1 primer, and Phusion DNA Polymerase. DNA is amplified with 8 cycles of PCR. After PCR, beads are separated on a magnet and the supernatant containing the PCR amplified library is transferred to a new tube, purified using the Zymo DNA Clean and Concentrator kit according to manufacturer's protocol and eluted in water. Purified HiChIP libraries were size selected to 300-700 basepairs using a double size selection with AMPure XP beads.
GEO Accession
GSM4217577