GSM7665218: 3-mt1-SUZ12; Homo sapiens; ChIP-Seq
Cells were collected, counted and washed once with PBS before crosslinking for 10 minutes with PBS containing 1 % formaldehyde (Sigma). Cells were crosslinked in 15 mL falcon tubes at a density of ~5x106 cells/mL. Crosslinking was quenched with 0.125 M Glycine prior to two PBS washes. Crosslinked cells were then snap-frozen on liquid nitrogen and stored at -80ºC. Crosslinked cells were thawed on ice and lysed in 5 mL of SDS-Lysis buffer (100 mM NaCl, 50 mM Tris pH 8.1, 5 mM EDTA pH 8.0, 0.5 % SDS, 1X protease inhibitor cocktail [Sigma; P8340]). Nuclei and chromatin were pelleted by centrifugation at 1200 rpm for 6 min at room temperature. The supernatant was then discarded, and the pellet was resuspended in 2 mL of ChIP buffer (33 mM Tris-HCl pH 8, 100 mM NaCl, 5 mM EDTA, 0.33% SDS, 1.67% Triton X-100, 1X protease inhibitor cocktail [Sigma; P8340]). Chromatin was sheared to approximately 200 bp to 500 bp fragments by sonication using a Bioruptor Plus (Diagenode) at high power. Total sonication time (i.e. cumulative “on” time) was 15min with 30 sec on/off pulses. Sonicated chromatin was incubated overnight with antibody while rotating at 4 ºC. Following ChIP experiments, the precipitated DNA was quantified using the Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Q32854). A total of 1-10 ng of DNA from each ChIP experiment was used for library preparation using the NEBNext Ultra II DNA Library Kit for Illumina (E7645) and NEBNext Multiplex Oligos for Illumina (Set#1 & #2; E7335 & E7500). Following adaptor ligation, DNA was PCR amplified for 4-10 cycles, depending on the amount of the input DNA. DNA purification was then performed using NEBNext Sample Purification Beads (E7767S). The quality and size distributions of DNA libraries were ascertained on a TapeStation (Agilent) using High Sensitivity D1000 ScreenTape assay reagents (Agilent; 5067-5585). The resulting libraries were then used for cluster generation and sequencing on an Illumina-based platform with a 150 bp paired-end read format (2-3x107 reads per sample).