title: GSM3535585: VSMC_Tri4C_ECAD9_2; Homo sapiens; OTHER
library_construction_protocol: Cells were fixed with 1% (v/v) formaldehyde in PBS for 10 min at room temperature. Fixation was quenched 0.2M glycene. Cells were washed with cold PBS twice and pelleted. Cell pellet was resuspended in a premixture of 250 µl cold lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630) and 50 µl protease inhibitors. Suspension was incubated on ice for 15 min and centrifuged. The pellet was washed once with 500 µl of cold lysis buffer, and carefully resuspended in 50 µl of 0.5% SDS. The suspension was then incubated in a 62 oC heating block for 7 min, followed by mixing with 145 µl water and 25 µl 10% Triton X-100, and incubated at 37 oC for 15 min. The suspension was mixed with 50 µl of buffer G, 120 U MboI (DpnII), 120 U Csp6I (CviQI), and 100 U Hin1II, and filled the total volume to 500 µl. For Single RE UMI-4C experiments, the suspension was mixed with 50 µl of buffer R, buffer B, and buffer G, respectively, for digestion with 100 U MboI, 100 U Csp6I, and 100 U Hin1II   in 500 µl final volume. All digestions were conducted at 37 oC overnight with rotation. The restriction enzymes were inactivated by incubating at 65 oC for 20 min. After cooling to room temperature, end blunting was performed by adding 3 µl of 10 mM dNTP, 8 µl of DNA polymerase I Klenow, and 4 µl of T4 DNA polymerase, and incubating at 37 oC for 1 hour with rotation. The suspension was then mixed with 460 µl water, 120 µl T4 ligase buffer, 100 µl 10% Triton X-100, 6 µl 20 mg/ml BSA, and 5 µl of 400 U/µl T4 ligase, and incubated for 4 hours at room temperature with rotation. The processed nuclei were pelleted and resuspended in 500 µl 1x T4 ligation buffer supplied with 50 µl 20 mg/ml proteinase K and 50 µl 10% SDS, and incubated at 55 oC for 30 min. For de-crosslinking, 60 µl 5M sodium chloride was added and the mixture was incubated at 68 oC for 2 hours. DNA was recovered using phenol-cholorform extraction.The re-arranged genomic DNA was sonicated to 300-400 bp fragments using Covaris S2 ultrasonicator. The fragmented DNA was double size-selected using 0.40x/1.0x of SPRI beads to remove reads below 100 bp and above 1000 bp, and eluted with a final volume of 70 µl of 10 mM Tris-HCl. The eluent was mixed with 10 µl 10x T4 ligation buffer , 10 µl 100 mM ATP, 5 µl 10 mM dNTP mix, 4 µl T4 DNA polymerase , 1 µl DNA polymerase Klenow, and 5 µl T4 PNK, and incubated for 30 min at room temperature. The repaired DNA was purified by using 1.0x SPRI beads, and eluted with a master mix of 94.5 µl 1x NEB buffer 2 and 0.5 µl 100 mM dATP. After removing the beads, 5 µl of Klenow exo- was added, and the mixture was incubated at 37 oC for 30 min for dA tailing. The processed DNA was purified by using 1.0x SPRI beads, and eluted with 20 µl 10 mM Tris-HCl. After dA-tailing, a custom Y shape adaptor was ligated to the fragments. For amplification, eight 100 µl reactions, each containing 400 µg preamplification library, 2 µM universal primer, 0.5 µM viewpoint-specific outer primer for each multiplexed viewpoint, and 1x SuperFi PCR master mix with 20% GC enhancer, was performed at following condition: 98 oC for 30s, 28 cycles of 98 oC for 10s, 62 oC for 10s, and 72 oC for 60s, and final extention at 72 oC for 5 min. The product was pooled and purified with 1.0x SPRI beads.
attributes.tag: GEO Accession
attributes.value: GSM3535585