GSM5986744: NTDChIP_Rep3_0hr; Homo sapiens; Mus musculus; ChIP-Seq
Native cChIP-seq was performed as described previously (Fursova et al. 2019). In brief, 5 x 107 ESCs were mixed with 2 x 107 Drosophila SG4 cells and nuclei were released by resuspending in RSB (10 mM Tris HCl pH 8, 10 mM NaCl, 3 mM MgCl2) with 0.1% NP40. Nuclei were pelleted at 1500xg for 5 min and then washed and resuspended in 1 ml MNase digestion buffer (RSB with 0.25 M Sucrose, 3 mM CaCl2, 1x PIC). Each sample was incubated with 200 units of MNase (Fermentas) at 37⁰C for 5 minutes, with gentle inversion. Digestion was stopped by addition of 4 mM EDTA. Following centrifugation at 1500xg for 5 min, the supernatant (S1 fraction) was retained and the remaining pellet was resuspended in 300 μl nucleosome release buffer (10 mM Tris HCl pH 7.5, 10 mM NaCl, 0.2 mM EDTA, 1x PIC), rotated at 4⁰C for 1 hr and then passed five times through a 27G needle using a 1 ml syringe. Following centrifugation at 1500xg for 5 min, the supernatant (S2) was combined with S1 fraction, aliquoted, snap frozen and stored at -80°C. For each IP, 100 μl S1/S2 nucleosomes were diluted to 1 ml total volume in native ChIP incubation buffer (70 mM NaCl, 10 mM Tris HCl, pH 7.5, 2 mM MgCl2, 2 mM EDTA, 0.1 % Triton-X100, 1x PIC) and immunoprecipitated with 3 µl H3K4me3 antibody (Klose Lab) overnight at 4⁰C. IPs were all set up in duplicate for each sample. 100 μl diluted chromatin was also set aside as an input sample. Protein A agarose beads (Repligen) were blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA in native ChIP incubation buffer, overnight at 4⁰C. 40 μl slurry of pre-blocked agarose beads were used to capture antibody-bound nucleosomes at 4⁰C for 1 hr. Beads were then washed 4x with Native ChIP wash buffer (20 mM Tris HCl, pH 7.5, 2 mM EDTA, 125 mM NaCl, 0.1 % Triton-X100, 1x PIC) and 1x TE buffer, pH 8. DNA was eluted by vortexing for 30 min in elution buffer (1% SDS and 0.1 M NaHCO3) and DNA was purified using a ChIP DNA Clean & Concentrator kit (Zymo Research). For each ChIP, DNA from the matched input control (10% of the IP) was also purified. Purified DNA was analysed using ChIP-qPCR.    For double cross-linked T7-SET1A ChIP and ZC3H4-T7 ChIP, 5 x 107 ESCs were fixed with 2 mM DSG (disuccinimidyl glutarate, Thermo Fisher Scientific) for 50 min at 25⁰C and then 1% formaldehyde (methanol-free, Thermo Fisher Scientific) for 10 min. Alternatively, for single cross-linked RNA Pol II ChIP, 5 x 107 ESCs were fixed with 1% formaldehyde for 10 min at 25⁰C. Fixation was quenched using glycine added to 125 mM. Cells were then pelleted at 1000xg for 5 min and washed with PBS. Cross-linked ESCs were mixed with 1 x 105 cross-linked HEK 293T/T7-SCC1 cells (1% formaldehyde, 15 min for SET1A ChIP; a gift from Martin Houlard, Nasmyth lab) or 2 x 106 cross-linked HEK 293T cells (1% formaldehyde, 10 min for RNA Pol II ChIP). Chromatin was prepared by incubation in 1 ml FA-lysis buffer (50 mM HEPES pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.5% NP-40, 0.1% Na-deoxycholate, 0.1% SDS, 1x PIC, 1 mM AEBSF. For RNA Pol II ChIP, EDTA concentration was increased to 2 mM and 10 mM NaF was added fresh) on ice for 10 min. Chromatin was sonicated using a BioRuptor Pico sonicator (Diagenode) at 4⁰C. Sonication was performed using 23-30 cycles of 30s on/30s off at full power, shearing genomic DNA to an average size of 0.5 kb. The sonicated material was pelleted at 20,000xg for 20 min, and the supernatant taken as sonicated chromatin. 300 μg chromatin was used per IP. Chromatin was diluted to 1 ml total volume per IP in FA-lysis buffer. An additional volume of diluted chromatin was taken to use as an input sample. Protein A agarose beads (Repligen) were blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA in 1x TE buffer at 4⁰C for 1 hr. Chromatin was pre-cleared with agarose beads (40 μl slurry beads per ChIP) at 4⁰C for 1 - 2 hr. The input sample was taken from the pre-cleared chromatin, and the remainder was immunoprecipitated overnight at 4⁰C with the appropriate amount of antibody: T7 (Cell Signalling, D9E1X, 10µl) or RNA Pol II N-terminal domain (Cell Signalling, D8L4Y, 15µl). Antibody-bound chromatin was precipitated for 3 hr at 4⁰C using 40 μl slurry of blocked Protein A agarose beads. Washes were carried out for 5 min each at 4⁰C, using FA-lysis buffer, FA-lysis buffer with 500 mM NaCl, 1x DOC buffer (10 mM Tris HCl, pH 8, 250 mM LiCl, 1 mM EDTA (2 mM EDTA for RNA Pol II ChIP), 0.5% NP-40, 0.5% Na-deoxycholate), and 2 washes with TE buffer (1x PIC and 1 mM AEBSF were added fresh to all wash buffers. 10 mM NaF was also added for RNA Pol II ChIP). DNA was eluted by vortexing for 30 min in elution buffer (1% SDS and 0.1 M NaHCO3). Cross-links were reversed for ChIPs and inputs at 65⁰C overnight with 200 mM NaCl and 2 μl RNase A (Sigma). Samples were then incubated with 20 μg Proteinase K for 1 hr at 45⁰C. DNA for ChIPs and inputs was purified using a ChIP DNA Clean & Concentrator kit (Zymo Research). Purified DNA was analysed using ChIP-qPCR. cChIP-seq libraries for both ChIP and Input samples were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina, following manufacturer's guidelines. Samples were indexed using NEBNext Multiplex Oligos. The average size and concentration of all libraries were analysed using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent) followed by qPCR quantification using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche). Libraries were sequenced as 40 bp paired-end reads in biological triplicate on Illumina NextSeq 500 platform.