GSM8566010: PNUTSdTAG_NTD_dTAG_rep3; Homo sapiens; Mus musculus; ChIP-Seq
5 x 10^7 ESCs were fixed with 1% formaldehyde (methanol-free, Thermo Fisher Scientific) 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 10^6 cross-linked HEK 293T(1% formaldehyde for 10 min). 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, 10 mM NaF) on ice for 10 min. Chromatin was sonicated using a BioRuptor Pico sonicator (Diagenode) at 4⁰C for 23 cycles (30s on/off) 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 respective antibody. 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 ice-cold 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 , 0.5% NP-40, 0.5% Na-deoxycholate), and 2 washes with TE buffer. DNA was eluted 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 were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina or NEBNext UltraExpress 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 quantification using Qubit HS DNA assay. Libraries were sequenced as 40 bp paired-end reads in at least biological triplicate on Illumina NextSeq 500 platform.