title: GSM2817554: 25.B replicate 2; Saccharomyces cerevisiae; Hi-C
library_construction_protocol: Cells were ground by mortar and pestle in liquid nitrogen for 10 minutes. Lysed cells were washed in 50 ml 1x NEBuffer 2 then centrifuged at 3000 rpm at 4°C for 5 minutes. The OD600 of the pellet was adjusted to 10.0 with 1x NEBuffer 2 followed by 2 volumes washes with 1x NEBuffer 2. Lysed cells were centrifuged at 4000 g at 4°C for 10 minutes and resuspended in 0.38 volume of the adjusted OD600=10.0 with 1x NEBuffer 2, SDS was added (0.1% final concentration) and the chromatin of mechanically lysed cells were incubated at 65˚C with shaking (950 rpm) for one hour. Triton X-100 was added at a final concentration of 0.95 % and incubated at 37˚C with shaking (950 rpm) for 10 minutes. Restriction digest was performed overnight at 37˚C with HindIII (NEB; 1500 units per sample).  The restriction sites were then filled in with Klenow (NEB) using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (all at a final concentration of 0.28 µM) for 60 minutes at 37˚C. Filled-in chromatin was mixed with 0.93 volumes of ligation reaction mix at a final concentration of 1x ligation buffer (NEB), 0.1mg/ml bovine serum albumin (BSA), 25 units / sample T4 DNA ligase (Life Technologies) for 6 hours at 16˚C. Reverse crosslinking (65˚C overnight in the presence of Proteinase K (Roche)) was followed by RNase A (Roche) treatment at 37 ˚C for 60 minutes and two sequential phenol/chloroform extractions. The DNA was precipitated in the presence of sodium acetate 3 M pH 5.2 (1/10 volume) and ethanol (2.5 x volumes) 45 min at -20˚C. The DNA extraction was repeated with one phenol extraction followed by two sequential phenol/chloroform extractions then one chloroform extraction. The DNA was precipitated in the presence of sodium acetate 3 M pH 5.2 (1/10 volume) and absolute ethanol (2.5 x volumes) overnight at -20˚C. After three consecutive washes with 70% v/v ethanol, the DNA concentration was determined using Quant-iT Pico Green (Life Technologies). Each 5 µg of Hi-C library DNA were incubated with  0.1 mg/ml BSA, 1x NEBuffer 2 (NEB), 0.2 mM dATP, and 15 units T4 DNA polymerase (NEB) in a total volume of 100 μl for 4 hours at 20˚C to remove any biotin from non-ligated fragment ends, followed by DNA purification (Qiagen PCR purification kit). DNA was eluted in 130 μl double distilled water.  Sonication was performed to obtain DNA fragments with a size peak around 400 bp (Covaris E220 settings: duty factor: 10%; peak incident power: 140W; cycles per burst: 200; time: 110 seconds). The sonicated DNA was end-repaired (T4 DNA polymerase, T4 DNA polynucleotide kinase, Klenow (all NEB)) for 30 minutes at 20˚C, followed by DNA purification (Qiagen PCR purification kit). dATP was added with Klenow exo- (NEB) for 30 minutes at 37˚C, and the enzyme was heat-inactivated (20 minutes at 65˚C). A double size selection using AMPure XP beads (Beckman Coulter) was performed: first, the ratio of AMPure XP beads solution volume to DNA sample volume was adjusted to 0.6:1. After incubation for 10 minutes at room temperature, the sample was transferred to a magnetic separator (DynaMag-2 magnet; Life Technologies), and the supernatant was transferred to a new Eppendorf tube. The ratio of AMPure XP beads solution volume to DNA sample volume was then adjusted to 0.9:1 final. After incubation for 10 minutes at room temperature, the sample was transferred to a magnet (DynaMag-2 magnet; Life Technologies). Following two washes with 70% v/v ethanol, the DNA was eluted in 300 µl of TLE (10 mM Tris-HCl pH 8.0; 0.1 mM EDTA). Biotin-marked ligation products were isolated using MyOne Streptavidin C1 Dynabeads (Life Technologies) on a DynaMag-2 magnet (Life Technologies) in binding buffer (10 mM Tris-HCl pH8, 1mM EDTA, 2 M NaCl) for 15 minutes at room temperature with rotation, followed by one wash in washing buffer (5 mM Tris-HCl pH8.0, 0.5 mM EDTA, 1 M NaCl), and one wash in ligation buffer (NEB). TruSeq indexed adapters (Illumina) were ligated onto Hi-C ligation products bound to streptavidin beads for 2 hours at room temperature (T4 DNA ligase NEB, in ligation buffer, slowly rotating). After washes in wash buffer (5 mM Tris, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20) and NEBuffer 2, the DNA-bound beads were resuspended in a final volume of 50µl NEBuffer 2. Bead-bound Hi-C DNA was amplified with 12 PCR amplification cycles using TruSeq primer PCR 1.0 and TruSeq primer PCR 2.0 primers (Illumina). The concentration and size distribution of Hi-C library DNA after PCR amplification was determined by Bioanalyzer profiles (Agilent Technologies). Hi-C libraries were sequenced using 50 bp paired-end sequencing on an Illumina HiSeq 2500 instrument in High Output mode, using Sequencing by Synthesis (SBS) v4 reagents.
attributes.tag: GEO Accession
attributes.value: GSM2817554