GSM3416314: HL60_S4_ATRA: ctrl_1_HiC; Homo sapiens; Hi-C
15x107 cells were crosslinked with Formaldehyde (2% FC, 10 min, RT), quenched with Glycine (125mM, 5 min, RT) and scraped from the plate. Cells were lysed on ice in lysis buffer (500 μl 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP-40 and 50 μl protease inhibitors, 15 min) and disrupted using a Dounce homogenizer (10X, 1 min on ice, 10X). Lysed cells were pelleted (4°C, 5,000rpm, 3 min), wash twice in 1X restriction enzyme buffer, suspend in 1X buffer and divided into five tubes with a final volume of 362μl. SDS was added (1% FC), incubated (65°C, 10 min), place back on ice and Quenched with Triton X-100 (10% FC). Chromatin was digested with HindIII (400U, 37OC, 16 h with rotation). Restriction site overhangs were filled in with biotin labeled Cytosine (1.5 μl 10 mM dATP, 1.5 μl 10 mM dGTP, 1.5 μl 10 mM dTTP, 37.5 μl 0.4 mM biotin-14-dCTP, and 50U Klenow), mixed carefully and incubated (45 min, 37°C). Tubes were placed on ice and the enzyme inactivated by SDS (10%), incubated (65°C, exactly 30 min) and placed back on ice immediately. Biotinylated fragments were ligated by adding 7.61 ml ligation mix (745 μl 10% Triton X-100, 745 μl 10x ligation buffer [500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 100 mM DTT], 80 μl 10 mg/ml BSA, 80 μl 100 mM ATP and 5.96 ml water), T4 DNA ligase (50U), mixed and incubated (4 h, 16°C). Crosslinks were reversed and protein degraded by Proteinase K (25 μl, 12 mg/ml) and incubation (16 h, 65°C). After 16 h, additional Proteinase K (25 μl 20 mg/ml) was added and further incubated (2 h, 65°C). The reactions were cooled to RT and DNA was purified by phenol (1X) and phenol:chloroform (1:1, 1X) extraction. The aqueous phase was transferred to high-speed centrifugation tubes (oakridge). The DNA was ethanol precipitated (2.5 volumes 100% Ethanol and 1/10th volume 3M Sodium Acetate), mixed by inversion, chilled (>30 min, -80°C), centrifuged (20 min, 10,000g, 4°C) and the DNA pellet dissolved in 450 μl 1x TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The DNA was phenol:chloroform (1:1) extracted twice more with centrifugation (5 min, 14,000rpm, RT). The DNA was ethanol precipitated (2.5 volumes 100% Ethanol and 1/10th volume 3M Sodium Acetate), mixed by inversion, chilled (>30 min, -80°C) and centrifuged (20 min, 10,000g, 4°C). The DNA pellet was washed with ice-cold 70% ethanol, centrifuged (10 min, >10,000g, 4°C), dried (~10 min) and suspended in 25 μl 1x TE. RNA was degraded by the addition of RNase A (1 μl 1 mg/ml), incubation (30 min, 37°C) and then the Hi-C libraries were pooled. Biotin was removed from unligated ends using T4 DNA polymerase. Mixed ~5μg of Hi-C library with T4 DNA polymerase reaction buffer (1 μl 10 mg/ml BSA, 10 μl 10x NEBuffer 2, 1 μl 10mM dATP, 1 μl 10mM dGTP) and T4 DNA polymerase (5U) in a total volume of 100 μl. Incubated the mixture (2 h, 12°C). Multiple 5 μg reactions were performed. Reactions were stopped by the addition of EDTA (2 μl 0.5M EDTA pH 8.0). Multiple 5 μg reactions were pooled and the DNA purified by three phenol pH 8.0:chloroform (1:1) extractions with centrifugation (5 min, 14,000 rpm, RT). The DNA was Ethanol precipitated (2.5 volumes 100% Ethanol and 1/10th volume 3M Sodium Acetate), mixed by inversion, chilled (>30 min, -80°C) and centrifuged (20 min, 10,000g, 4°C). The DNA pellet was washed with 70% Ethanol, dried, suspended and pooled in a total volume of 100 μl H2O. The DNA was sheared to a size of 300-500bp using a Covaris S2 instrument (duty cycle 5, intensity 5, cycles/burst 200, time 60 secs for 4 cycles). The sheared DNA ends were repaired (14 μl 10x ligation buffer, 14 μl 2.5 mM dNTP mix, 5 μl T4 DNA polymerase, 5 μl T4 polynucleotide kinase, 1 μl Klenow DNA polymerase and 1μl H2O) and incubated (30 min, RT). DNA was purified using a Qiagen MinElute column according to manufacturer's recommendations and the DNA eluted twice with Tris-Low-EDTA (TLE: 15 μl 1x [10 mM Tris pH 8.0, 0.1 mM EDTA]). An 'A' tail was added to the DNA fragments using Klenow (5 μl [10x NEBuffer2, 10 μl 1 mM dATP, 2 μl H2O and 3 μl Klenow (exo-)]) and incubation (30 min, 37°C). The Klenow was inactivated (20 min, 65°C) and the reaction was cooled on ice. The reaction volume was reduced to 20 μl using a speedvac, the DNA loaded on a 1.5% agarose gel with 1X TAE, run for 3 hours at 80V and DNA fragments between 300 and 500 bp were excised. The DNA was purified using a Qiagen gel extraction kit according to manufacturers recommendations and the DNA eluted with 50 μl 1x TLE. Combined the purified DNA in a final volume of 300 μl 1x TLE and determined the DNA concentration by QuBit. To enrich for biotin-labeled fragments magnetic streptavidin beads (100 μl) were washed twice with Tween Buffer (TB: 400 μl [5 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween]) and suspended in a volume of 2x No Tween Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 M NaCl) equivalent to the remaining Hi-C library volume. All subsequent steps were performed in DNA LoBind tubes. Combined the beads with Hi-C DNA and incubated (15 min, RT, slow rotation). Placed the tubes on a magnetic particle concentrator, removed the supernatant, washed the beads in NTB (NTB: 400 μl [5 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 1 M NaCl]) and then with ligation buffer (100 μl 1x). Suspend the beads in ligation buffer (30 μl 1x) and transferred to a new tube. The DNA bound to the streptavidin beads (~100 ng) was used for sequencing library preparation according to Illumina instructions. Resuspention buffer (2.5 µl), DNA adaptor index (2.5 µl) and Ligation Mix 2 (2.5 µl) were added to the tube, and incubated (30°C, 2 h). To stop the reaction Stop Ligation buffer (5 µl) was added to the tube. Removed non-ligated adapters by placing the tube on a magnetic particle concentrator and washing the beads twice with TB (400 μl 1x), once with NTB (200 μl 1x) and twice with NEBuffer 2 (200 μl and 50 μl 1x). Suspended the beads in 50 μl 1x NEBuffer 2 and transfered to a new tube. Determined the number of PCR cycles necessary to generate enough product for sequencing; set up four PCR reactions with 6, 9, 12 or 15 cycles (9 µl of sample [8 µl 1x NEBuffer2 + 1 µl DNA bound beads], 9 µl Enhancer PCR mix and 2 µl PCR cocktail. Run PCR: 95°C – 3 min, {98°C – 20 sec, 60°C – 15 sec, 6-15 cycles 72°C – 30 sec} 6-15 cycles, 72°C – 5 min, 4°C – forever. Determined the optimal cycle number by running the PCR reactions on a 5% polyacrylamide gel (60 min, 80 V). Amplified the remainder of the Hi-C-library-bound streptavidin beads in a large-scale PCR with the optimal number of PCR cycles. Pooled the PCR products from the separate wells and reclaimed the beads. Kept 1% of the large-scale PCR product separate to run on a gel and purify the remainder of the PCR product with Ampure beads (1.8x volume) according to the manufacturer's recommendations. Eluted the DNA with TLE (50 μl 1x) and compared 1% of the Ampure bead purified PCR product to the 1% aliquot of original PCR product on a 5% polyacrylamide gel to insure the successful removal of the PCR primers. Sequenced the Hi-C library with Illumina paired end sequencing (50 bp). Illumina TruSeq® Nano DNA LT Sample Preparation Kit