Truseq indexed adapter

A total of 1 µg amplified cDNA was barcoded with a unique set of standard Illumina TruSeq indexed adapters by ligation and sequenced on an Illumina HiSeq 2000 high-throughput sequencer according to manufacturer’s protocol at the department of Clinical Genetics, Amsterdam UMC Location VUmc in Amsterdam, The Netherlands. Paired-end reads of 2 × 100 bp were trimmed using Trimmomatic (v0.32, https://github.com/timflutre/trimmomatic) [74 (link)] (reads ≥ 15 nucleotides passed the filter) and aligned to the hg19 human reference genome using TopHat/Bowtie2 (v2.1.0, https://ccb.jhu.edu/software/tophat/index.shtml) [75 (link)]. We recovered 31,370,153 ± 10,903,535 uniquely aligned paired-end sequence reads per sample as generated by samtools (v0.1.19, http://www.htslib.org/) spanning 19,250 ± 1865 known transcripts.

DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, D) according to the manufacturer’s protocol from segregant plant pools, and quantified using the Qubit dsDNA BR assay kit. Illumina sequencing library preparation and bisulfite conversion was conducted as described in Kawakatsu [55 (link)]. Briefly, DNA was End Repaired using the End-It kit (Epicentre, Madison, WI), A-tailed using dA-Tailing buffer and 3μL Klenow (3’ to 5’ exo minus) (NEB, Ipswich, MA) and Truseq Indexed Adapters (Illumina, San Diego, CA) were ligated overnight. Bead purified DNA was quantified using the Qubit dsDNA BR assay kit, and stored at -20°C. At least 450ng of adapter ligated DNA was taken into bisulfite conversion which was performed according to the protocol provided with the MethylCode Bisulfite Conversion kit (Thermo Fisher, Waltham, MA). Cleaned, converted single stranded DNA was amplified by PCR using the KAPA U+ 2x Readymix (Roche Holding AG, Basel, CH): 2 min at 95°C, 30 sec at 98°C [15 sec at 98°C, 30 sec at 60°C, and 4 min at 72°C] x 9, 10 min at 72°C, hold at 4°C. After amplification, the DNA was bead purified, the concentration was assessed using the Qubit dsDNA BR assay kit, and the samples were stored at -20°C. WGBS library was sequenced as part of a large multiplexed pool paired-end 150 bp on an Illumina HiSeq 4000.

ChIP-seq experiments were performed as previously described^{40 (link)}. Briefly, cells were fixed with 1% formaldehyde at RT for 10 min, lysed and sonicated in RIPA buffer containing 0.2% SDS (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.2% SDS, 0.5 mM PMSF, and 1x Protease inhibitor cocktail (Roche). Chromatin was cleared by centrifugation at 20,000×g for 10 min and incubated with 2–10 μg of antibodies pre-bound to Dynabeads Protein G (Life technologies). The antibodies used here were specific for STAG1 (Abcam, ab4457, lot: GR279696-4), STAG2 (Abcam, ab4464, Lot: GR271549-1), SMC1A (Bethyl, A300-055A, lot 5), and CTCF (Cell Signaling Tech, 2899 s, lot 2). Purified ChIP DNA was end-repaired, end adenylated, and ligated with Illumina Truseq indexed adapters. The ligated DNA was purified with AMPure XP beads (Beckman Coulter) and amplified with KAPA HiFi DNA Polymerase (KAPA Biosystems) for 8 to 13 cycles. After amplification, the library DNA was size-selected with AMPurex XP beads to 200–600 bp range, and the purified libraries were multiplexed for sequencing at the Center for Computational and Integrative Biology (CCIB) DNA Core at MGH.

100 ng genomic DNA from MEFs or tissues was treated with bisulfite using EpiTect Fast Bisulfite Conversion Kits (Qiagen). PCR was performed using primers specific for CGIs in the promoter region of Uncx gene with the bisulfite converted DNA as template (forward primer 5'-TGATGTTGATAAAGTAA AGY(C/T)G-3', reverse primer 5'-CTCCAACCTACCTACAAACTTAAA-3'). The PCR products (408 bps) were further purified and ligated to Illumina Truseq indexed adapter (1:30 dilution). The libraries generated from each PCR product were mixed at equal molar concentration and the sequencing was performed on a MiSeq instrument using MiSeq Reagent Kits v2 (Illumina, 2 × 250 cycles).