title: sequencing for HiC: L3
design: We constructed Hi-C libraries, using the original sample as input. After grinding with liquid nitrogen, the sample was cross-linked by 4% formaldehyde solution at room temperature in a vacuum for 30 mins. 2.5 M glycine was added to quench the crosslinking reaction for 5 min and then put it on ice for 15 min. The sample was centrifuged at 2500 rpm at 4C for 10 mins, and the pellet was washed with 500 L PBS and then centrifuged for 5 min at 2500 rpm. The pellet was resuspended with 20 L of lysis buffer (1 M Tris-HCl, pH 8, 1 M NaCl, 10% CA-630, and 13 units protease inhibitor), the supernatant was then centrifuged at 5000 rpm at room temperature for 10min, The pellet was washed twice in 100 L ice cold 1x NEB buffer and then centrifuged for 5 min at 5000 rpm. The nuclei were re-suspended by 100 L NEB buffer and solubilized with dilute SDS followed by incubation at 6C for 10 min, After quenching the SDS by Triton X-100, an overnight digestion was applied to the samples with a 4-cutter restriction enzyme Mbol (Dpnll) (400 units) at 37C on a rocking platform. The following steps involved marking the DNA ends with biotin-14-dCTP and blunt-end ligation of the cross-linked fragments. The proximal chromatin DNA was re-ligated by ligation enzyme. The nuclear complexes were revers cross-linked by incubation with the proteinase K at 65C. DNA was purified by the phenol-chloroform extraction. Biotin was removed from non-ligated fragment ends using T4 DNA polymerase. Ends of sheared fragments by sonication (200-600 base pairs) were repaired by the mixture of T4 DNA polymerase, T4 polynucleotide kinase and Klenow DNA polymerase. Biotin-labeled Hi-C samples were specifically enriched using streptavidin Cl magnetic beads. After adding A-tails to the fragment ends and following ligation by the illumina paired-end (PE) sequencing adapters, Hi-C sequencing libraries were amplified by PCR (12-14 cycles) and sequenced on lllumina platform.
library_name: clamL3