Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: ChIP assays were performed as previously described (Lee et al., 2006) . Briefly, 2-5x10^7 cells were crosslinked with 1% paraformaldehyde (Electron microscropy science #15710) at room temperature for 10 min and sonicated to generate chromatin fragments of 200-600bp. The sheared chromatin was immunoprecipated with a specific antibody. Total RNA was extracted with Trizol (Invitrogen #15596), treated with DNAase I (NEB #M0303) for 20 min at room temperature and purified with RNeasy Mini kit (Qiagen #74106).Circular Chromosome Conformation Capture (4C) technique was performed as described (van de Werken et al., 2012, Methods Enzymol). Cross-linked chromatin was digested with HindIII, ligated under diluted conditions. Cross links were reversed and DNA was further digested by NlaIII and again ligated under diluted conditions.For each viewpoint approximately 3.2 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina adaptor sequence. Multiplexed samples were sequenced on the Illumina Nextseq500 system using 75 bp single-end reads according to the manufacturer's specifications. 4C-seq reads were sorted, aligned, and translated to restriction fragments using the 4C-seq pipeline according to (van de Werken et al., 2012, Methods Enzymol). ChIP-seq and RNA-seq libraries were constructed by following instrucitons in KAPA HTP Library Preparation Kits (Kapa Biosystems, #KK8234) and TruSeq Stranded Total RNA Library Prep kits with Ribo-Zero Human/Mouse/Rat (Illumina, #RS-122-2201 and #RS-122-2202), respectively. Indexed primers for ChIP-seq libraries are from Bioo Scientific (#514104 ). For 4C-seq, Cross-linked chromatin was digested with HindIII, ligated under diluted conditions. Cross links were reversed and DNA was further digested by NlaIII and again ligated under diluted conditions. For each viewpoint, approximately 3.2 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina adaptor sequence. Multiplexed samples were sequenced on the Illumina Nextseq500 system using 75 bp single-end reads according to the manufacturer's specifications.