ChIP-seq

Chromatin immunoprecipitation (ChIP) was performed as previously described (Letting et al., 2004). Antibodies include: CTCF (Millipore; 07-729), Rad21 (Bethyl Laboratories; A302-583A), mCherry (Abcam; {"type":"entrez-nucleotide","attrs":{"text":"Ab167453","term_id":"45421876","term_text":"AB167453"}}Ab167453), RNAPII (Cell Signaling; Cat#14958), IgG from rabbit serum (Sigma; 15006). Quantitative polymerase chain reaction (qPCR) was performed using Power SYBR Green kit (Invitrogen; 4368577) with signals detected by ViiA7 System (Life Technologies). ChIP-seq libraries were prepared using Illumina’s TruSeq ChIP sample preparation kit (Illumina, Cat#IP-202-1012) according to manufacturer’s specifications, with the addition of size selection (left side at 0.9x, right side at 0.6x) using SPRIselect beads (Beckman Coulter, Cat#{"type":"entrez-nucleotide","attrs":{"text":"B23318","term_id":"2508949","term_text":"B23318"}}B23318). Library size was determined (average 351 bp, range 333-372 bp) using the Agilent Bioanalyzer 2100, followed by quantitation using real-time PCR using the KAPA Library Quant Kit for Illumina (KAPA Biosystems; Cat#KK4835). Libraries were then pooled and sequenced (1x75bp) on the Illumina NextSeq 500 platform according to manufacturer’s instructions. Bclfastq2 v 2.15.04 (default parameters) was used to convert reads to fastq.