GSM9574247: WT_Tm_H3K27ac_HiChIP_SET2_rep1; Homo sapiens; OTHER
Cells were fixed with 1% formaldehyde for 10 min at room temperature and quenched using 0.125 M glycine for 5 min, followed by washing with cold PBS. After cell lysis, cross-linked chromatin was digested with the DpnII restriction enzyme, with enzyme amounts adjusted in proportion to the number of cells, followed by fill-in with biotin-14-dATP (Jena Bioscience) and proximity ligation. The resulting ligation products were sheared by sonication using a Qsonica sonicator (30 s ON/30 s OFF, 10 cycles) and incubated overnight at 4 ℃ with either an H3K27ac antibody (Abcam, ab4729) or an ATF4 antibody (Cell Signaling Technology, 11815). After sequential washes with standard ChIP wash buffers, chromatin–antibody complexes were reverse cross-linked overnight. De-cross-linked DNA was purified and quantified using Qubit dsDNA HS Assay Kit (Invitrogen, Q32854) and 5 ng of DNA was subjected to biotin pull-down using a streptavidin C-1 magnetic beads. Tagmentation was directly performed on bead-bound DNA with Tn5 transposase with enzyme amounts adjusted according to the DNA input, followed by washing with appropriate buffers. Tagmented DNA was released from the beads by a simplified PCR procedure and further amplified using Nextera PCR primers. Amplified libraries were size-selected and purified using AMPure XP beads (0.5x–0.75x).