Biotinylated dna oligonucleotides

NG Capture-C was performed as described22 (link). Cells were obtained using the fetal liver culture system outlined above. 3C libraries were made using standard methods similar to the protocol for in situ Hi-C. Prior to oligonucleotide capture, 3C libraries were sonicated to 200 bp and Illumina paired-end sequencing adaptors (NEB E6040 / E7335 / E7500; Agilent Herculase II) were added. Samples were indexed, allowing multiple samples to be pooled prior to oligonucleotide capture using biotinylated DNA oligonucleotides designed for the Hba-a1&2, Hbb-b1&2 and Slc25A37 promoters (Sigma). The first hybridisation reaction was scaled up relative to the number of samples included in the reaction to maintain library complexity (Nimblegen Roch SeqCap EZ). Following a 72h hybridisation step a streptavidin bead pull down (Invitrogen M270) was performed followed by multiple bead washes (Nimblegen SeqCap EZ) and PCR amplification of the captured material (Kappa / Nimblegen SeqCap EZ accessory kit v2). A single volume, double capture step was performed. The material was sequenced using the Illumina MiSeq with 150bp PE reads (300bp V2 chemistry). Data were analysed using analysis scripts (available via Github) and R was used to normalize data and generate differential tracks.