GSM7106324: KO input; Mus musculus; ChIP-Seq
ChIP assays were performed using the SimpleChIP Plus Enzymatic Chromatin IP Kit (CST, MA, USA, #9005) according to the procedures provided by the manufacturer. Liver tissues were harvested at the indicated time and quickly cut into ~0.2 cm3 pieces, crosslinked with 1% formaldehyde for 15 min and quenched with 2.5 M glycine for 5 min at room temperature. After centrifugation, the pieces were homogenized in ice-cold PBS first with a loose pestle and then with a tight pestle, permeabilized and re-suspended in micrococcal nuclease (MNase) buffer and incubated with MNase for 20 min at 37°C. The MNase-digested chromatin was centrifuged at 14,000 rpm for 1 min. Afterwards, the cells were resuspended in ChIP buffer and the chromatin was sonicated on ice (30 s on/30 s off) to obtain soluble sheared chromatin (average DNA length of 200–500 bp). Approximately 10 µg of chromatin was diluted in ChIP buffer and pre-cleared with Dynabeads Protein G (Invitrogen, 10004D) for 2 h at 4°C. Anti-H3K27ac antibodies (5 µg, Abcam, Cambridge, UK, ab4729) and anti-MeCP2 antibodies (8 µg, Abcam, Cambridge, UK, ab2828) were added to the pre-cleared chromatin, followed by rotation overnight at 4°C. The Dynabeads were washed with low/high salt solutions and eluted twice in TE buffer containing 1% SDS at 65°C for 15 min. The combined eluates were isolated by reversal of cross-linking, incubated with RNase A (10 µg/ml) followed by proteinase K (0.1 mg/ml), and the DNA was extracted using a phenol-chloroform extraction protocol. DNA quality was assessed using an Agilent (Santa Clara, CA, USA) bioanalyzer and quantified using a Qubit fluorometer (Invitrogen). Sequencing libraries were constructed from DNA samples (ChIP and control samples) with the Illumina TruSeq V3 library construction protocol (Illumina, San Diego, USA). Sequencing runs were performed on an Illumina HiSeq 2000 for 51 cycles in single-end mode using Illumina TruSeq V3 chemistry (flowcells C56J3ACXX and C56KBACXX), or on an Illumina NextSeq 500 for 76 cycles in single-end mode using NextSeq V4 chemistry (flowcells H0Y1PBGXX, H11P6BGXX, and H13V2BGXX). ChIP-seq library preparation of ChIP DNA or input DNA was performed using TruSeq ChIP Sample Prep Kit (Illumina). Library DNA was quantified using KAPA Library Quantification Kit (Complete Kit (Universal) for Illumina platforms) (Kapa Biosystems) and library average size was determined by Agilent DNA 1000 Kit (Agilent Technologies) run on a 2100 Bioanalyser System (Agilent Technologies). Libraries were pooled for Single-Read 50bp sequencing on an Illumina HiSeq 2500 or 4000 platforms.