GSM2536181: N2 (Hi-C); Caenorhabditis elegans; Hi-C
DNA was purified as in Rao et al. (2014) and sheared in a 130 µl microTUBE (Covaris, 520045) using an S2 Covaris (Covaris 520045) for 55s with duty cycle 10%, intensity 4, and 200 cycles/burst. 300-500bp fragments were selected as in Rao et al. (2014), except that 40 µl of AMPure XP beads were used for the second size selection step. Biotinylated fragments were pulled down according to Rao et al. (2014) and Dynabeads were washed in 100 ul 1X End-It DNA End-Repair Kit (Lucigen, ER81050) End-Repair buffer. Beads were resuspended in 100 µl of End-Repair Mix (10 µl 10X End-Repair buffer, 10 µl 2.4mM dNTP mix, 10 µl 10mM ATP, 2 µl End-Repair enzyme mix, 68 µl water) and incubated at room temperature for 45 min with gentle rotation. A-tailing and adapter ligation were performed as in Rao et al. (2014) using 2 µl NEXTflex DNA Barcode (Bioo Scientific, 514101). To avoid PCR inhibition, the beads were divided between seven 50 µl PCRs and amplified for 4-6 cycles using Phusion polymerase (Thermo Fischer Scientific, F-530L) and NEXTflex Primer Mix (Bioo Scientific, 514107-48). The final library was purified according to Rao et al. (2014). To verify successful Hi-C, 1 µl of the library was cloned using a Zero Blunt TOPO PCR cloning kit (Life Technologies, 450245), and 20-50 colonies were amplified and sequenced. If at least 50% of the fragments contained informative ligations, the library was sequenced using 100 bp paired end reads on a HiSeq4000 platform. A step-by-step version of this protocol will be available on our website.