title: GSM8059889: EZH2-pos-iPS-ConditionL; Homo sapiens; ChIP-Seq
library_construction_protocol: Mouse ESC, K562 or human iPS 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 before proceeding with the rest of the ChIP assay. rChIP Condition L - ChIP conditions resulting in RNase-dependent loss of PRC2. ChIP assay with RNAse A treatment was performed based on a previous study [Long et al 2020]. Specifically, crosslinked cells were thawed on ice, lysed in 2 mL lysis buffer (50 mM Tris-Cl pH 8.1, 10 mM EDTA, 0.5-1.0 % SDS, plus 1X protease inhibitor cocktail [Sigma; P8340]) and incubated on ice for 10 mins. Chromatin was sheared to approximately 200bp-500bp fragments by sonication using a Bioruptor Plus (Diagenode) at high power. Total sonication time (e.g. total “on” time) was 20 mins with 30 sec on/off pulses. After sonication, the lysate was cleared by centrifugation at 16,300 g for 10 min at 4 ºC. The supernatant was stored at -80ºC or used directly in the ChIP assay. Lysate was diluted 1:5 in IP buffer (16.7 mM Tris-HCl pH 8.1, 1.2 mM EDTA, 167 mM NaCl, 1% Triton X-100 and 1X protease inhibitor cocktail [Sigma; P8340]) and pre-cleared with 50 µL protein G Dynabeads (ThermoFisher; 10004D) for 1 hour at 4 ºC. The sonicated lysate was incubated overnight with antibody while rotating at 4 ºC. Prior to overnight incubation with the antibodies, an input sample was taken (1%). RNase A (DNase and protease-free Thermo Fisher Scientific; #EN0531) was added along with the antibody to the plus RNase A samples at a final concentration of 50 µg/mL. Extracts from 2.5x106 cells were used per ChIP (+/- RNase A). The Next morning, Protein G Dynabeads were equilibrated in IP buffer: 25 µL beads were added to each ChIP and incubated for 1 hour at 4ºC. After incubation, beads were washed twice in low salt buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.1 % SDS, 1 % Triton x-100), twice with high salt buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 500 mM NaCl, 0.1 % SDS, 1% Triton x-100), twice in LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 1% Sodium Deoxycholate (w/v), 1% NP-40 substitute) followed by one final wash in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). All the washes were performed at 4 ºC. Immunoprecipitated material was eluted from the beads with 120 µL elution buffer (0.1 M NaHCO3, 1% SDS) for 20 min at room temperature. The supernatant was retained and 200 mM NaCl was added and incubated along with input samples overnight at 65 ºC while shaking to reverse the crosslinks. The eluted material was then subjected to RNase A (DNase and protease-free Thermo Fisher Scientific; #EN0531) and Proteinase K (ThermoFisher; #EO0491) treatments prior to DNA clean up (QIAGEN MinElute PCR Purification Kit Cat#28004). ChIP enrichments were analysed by qPCR using the QuantiNova SYBR Green PCR kit (Qiagen; 208054). rChIP Condition M - ChIP conditions resulting in no changes to the PRC2 occupancy following RNase A treatment. 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. Prior to overnight incubation with antibodies, an input sample was taken (1 %). RNase A (DNase and protease-free Thermo Fisher Scientific; #EN0531) was added along with the antibody to the plus RNase A samples at a final concentration of 50 µg/mL. Extract from 2.5x106 cells were used per ChIP (+/- RNase A). The following morning, samples were clarified by centrifugation at 20,000 g for 20 min at 4 ºC. Following clarification, the chromatin was incubated for 3 hours with Protein G Dynabeads (ThermoFisher; 10004D; 50 µL beads were used per ChIP). After incubation, the beads were washed three times in Mixed Micelle Buffer (150 mM NaCl, 20 mM Tris pH 8.1, 5 mM EDTA pH 8.0, 5.2 % Sucrose, 1 % Triton X-100, 0.2 % SDS), twice with Buffer 500 (0.1 % Sodium Deoxycholate, 1 mM EDTA pH 8.0, 50 mM HEPES pH 7.5, 1 % TritonX-100), twice with LiCl detergent wash (0.5 % Sodium Deoxycholate, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5 % NP-40,10 mM Tris pH 8.0) and finally, one wash with TE. All the washes were performed at 4ºC. Immunoprecipitated material was eluted from the beads with 100 µL elution buffer (0.1 M NaHCO3, 1 % SDS) while shaking for 1 hour at 65 ºC. The supernatant was retained and incubated overnight at 65 ºC while shaking to reverse the crosslinks. The eluted material was then subjected to RNase A (DNase and protease-free Thermo Fisher Scientific; #EN0531) and Proteinase K (ThermoFisher; #EO0491) treatments prior to DNA clean up (QIAGEN MinElute PCR Purification Kit Cat#28004). ChIP enrichments were analysed by qPCR using the QuantiNova SYBR Green PCR kit (Qiagen; 208054). Following ChIP (+/- RNase A) 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) (GeneWiz/Azenta)