GSM5605659: RNA-seq_BxPC3_2; Homo sapiens; RNA-Seq
We used 40ml 2% formaldehyde solution to crosslink the tissue for 15mins at room temperature in vacuum. Then, we added 4.324ml 2.5M Gly to quench the cross-linking reaction. Remove the supernatant and tissues were in the precipitate. We kept the tissue in liquid nitrogen.  Tissues can be resuspended by 25ml extraction buffer (10 mM Tris-HCl at pH 8, 10 mM MgCl2,0.4 M sucrose, 0.1 mM phenylmethylsulfonyl fluoride [PMSF], 5 mM b-mercaptoethanol, and 13 protease inhibitor; Roche). Next filtered by miracloth (Calbiochem).  We then did the centrifugation at 4000rpm at 4℃ for 20mins. After that, resuspended the pellet by 1ml extraction II buffer (10 mM Tris-HCl pH8, 1% Triton X-100, 0.1mM PMSF, 0.25 M sucrose, 10mM MgCl2, 5mMb mercaptoethanol, 13 protease inhibitor). Later, the solution was centrifuged for 10 mins at 14000 rpm in 4℃. Resuspended the pellet by 300μl extraction buffer III (10mM Tris-HCl pH8, 1.7M sucrose, 2mM MgCl2, 1μL protease inhibitor, 0.1mM PMSF, 5mM b-mercaptoethanol, 0.15% Triton X-100). Afterwards, load another 300μl clean extraction buffer III and centrifuged at 14000rpm for 10mins. Removed the supernatant and then washed the pallet by 500ul ice-cold 1x CutSmart buffer, centrifuged at 2500g for 5mins each time. The remaining pallets were the nuclei. Hereafter, we used the restriction enzyme buffer to wash the pallet twice and moved to a safe-lock tube.  The next step has solubilized the chromatin with dilute SDS and incubated at 65℃ by 10mins. Then quenched SDS by Triton X-100 Overnight. Next, the nuclei were digested by 4 cutter restriction enzyme (400 units MboI) at 37℃ on a rocking platform. The next step was marked the DNA ends by biotin-14-dCTP and then did the blunt-end ligation between cross-linked fragments. Thus, the ligation enzyme could ligate the proximal chromatin DNA. We incubated the nuclear complexes with proteinase K at 65℃ and then the nuclear complexes were reversed cross-linked.  DNA was then purified by phenol-chloroform extraction. We used T4 DNA polymerase to remove the Biotin-C from non-ligated fragments. Next, we used sonication to shear the fragments to 200-600 base pairs and repair the fragments by the mixture of T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase. Biotin-labeled DNA fragments were then enriched by streptavidin C1 magnetic beads. The Hi-C library from the beads was sequenced on the Illumina HiSeq X Ten platform with 150 bp paired-end reads.