alias: GSM2046419
title: GSM2046419: 3D rep 1; Homo sapiens; OTHER
library_construction_protocol: The methods for Hi-C library construction were adopted from (Belton JM, McCord RP, Gibcus JH, Naumova N, Zhan Y, Dekker J. Hi-C: a comprehensive technique to capture the conformation of genomes. Methods 2012). For each Hi-C library, ~20*106 cells were resuspended in 1 mL ice-cold lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 0.2% Igpel, 10 mL protease inhibitor cocktail) and incubated on ice for 15 min. Cells were homogenized on ice. Cells were collected by spinning for 5 minutes at 2,000 xg, then washed twice in 500 mL of ice cold 1x NEB buffer 2. Cells were distributed between 4 centrifuge tubes. Chromatins in each tube were digested with 400u of restriction enzyme HindIII in 1x NEB buffer 2 at 37 C overnight on a spin wheel. After HindIII digestion, restriction site overhanging ends were filled and labeled with biotin with DNA polymerase I large (Klenow) fragments in a reaction containing dATP, dGTP, dTTP, and biotin-14-dCTP in each of the 4 Hind III digestion tube. DNA fragments labeled biotin-14-dCTP from each of the 4 tubes were ligated at 16 C for 4 hours in an 8.23 mL reaction containing 1x ligation buffer, 1% Triton X-100, 1 mg/ml Bovine serum albumin (BSA),10 mM ATP, and 50u T4 DNA ligase . Reverse cross-linking started with 50 ml of 10 mg/ml proteinase K added to each tube and incubated at 65 C for 4 hours. Then, another 50 ml of proteinase K were added to each tube and continued incubating at 65 C overnight. Next, DNA was extracted with saturated phenolchloroform (1:1) and desalted by using AMICON! Ultra Centrifugal Filter with 1 x TE buffer. The final volume of desalted DNA was adjusted to 100 mL in 1 x TE buffer. Removal of Biotin from un-ligated ends was carried out in 8 individual reactions each of 50 mL containing 5 mg of Hi-C 405 DNA, 1 mg/ml BSA,1X NEB buffer 2, 25 nM dATP, 25 nM dGTP, and 15u T4 DNA polymerase at 20 C for 4 hours. The Hi-C DNA was then pooled in a single tube, purified with single phenol extraction, and precipitated by ethanol. The DNA was re-dissolved in 105 ml of water, and transferred to a microTUBE AFA tube. DNA fragmentation was performed in a Sonicator. The DNA fragments of 200 – 400 bp were recovered with Agencourt AMPure XP mixture. DNA fragment ends were repaired in a 70 mL reaction containing 1X ligation buffer, 0.25 mM of dNTP mixture, 7.5 u of T4 DNA polymerase, 25u of T4 polynucleotide kinase, 2.5u of DNA polymerase I large fragment at 20 C for 30 min. The reaction was purified with a MinElute column. The DNA was eluted in 32 mL of elusion buffer for A-tailing, which was performed in a 50 mL reaction containing 5 mg DNA, 1X NEB buffer 2, 0.2 mM dATP, 15u Klenow fragment. The reaction was incubated at 37 C for 30 min, then at 65 C for 20 min. For Streptavidin pull-down, the biotinylated Hi-C products were mixed with MyOne C1 streptavidin bead solution. Non-specifically binding DNA was removed by washing with 1X binding buffer (5 mM Tris-HCl (pH8.0), 0.5 mM EDTA, and 435 1 M NaCl, then with 1X T4 Ligation buffer). The DNA-bound beads were resuspended in 38.75 ml of 1 X ligation buffer for adapter ligation. Illumina adapter ligation was performed in a 50 mL reaction by adding to the DNA-bound beads suspension of 1X T4 ligation buffer, 90 pM of Illumina paired end adapter, 3u of T4 DNA ligase. The reaction was incubated at room temperature for 2 hours. The beads were reclaimed, and the supernatant discarded. The beads were washed twice in Tween Wash Buffer (5 mM Tris-HCl pH8.0, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween 20), once in 1X binding buffer (5 mM Tris-HCl (pH8.0), 0.5 mM EDTA, and 1 M NaCl, and twice in 1 X NEB buffer 2. After the last wash, the beads were resuspended in 20 ml of 1X NEB buffer 2. The Hi-C DNA sample was amplified by 15 PCR cycles for Illumina HiSeq sequencing. Each PCR reaction in 25 ml, 1.5 ml of Bead-bound Hi-C DNA, 0.35 ml of PE primer 1.0, 0.35 ml of PE primer 2.0, 0.2 ml of 25 mM dNTP, 2.5 ml of 10X PfuUltra buffer, 19.6 ml of H2O, and 0.5 ml of PfuUltra Fusion DNA polymerase. The PCR cycling parameters were 98 C for 30 seconds, followed by 15 cycles at 98 C for 10 seconds, 65 C for 30 seconds, and 72 C for 30 seconds, and a final extension at 72 C for 7 minutes. PCR products pooled from the supernatant of multiple reactions were subjected to AMpure XP beads purification to remove primer dimers. Each Hi-C library that passed the QC procedure was then sequenced in a single lane of a flow cell on a HiSeq 2000 sequencer to generate paired-end sequence reads at 100 bases per end read.
identifiers.namespace: GEO
identifiers.id: GSM2046419