title: GSM2417292: cdc20 release40; Saccharomyces cerevisiae; Hi-C
library_construction_protocol: For each Hi-C experiment aliquots of 1-3 x 10e9 cells in 150 ml YPD/SC were fixed in 3% formaldehyde (Sigma) for 20 min at room temperature and quenched with 25 ml glycine 2.5 M for 20 min at 4°C. Cross-linked cells were recovered through centrifugation, washed with YPD and incubated for 30 min in 10 ml of sorbitol 1M, DTT 5mM and Zymolyase 100T (CFinal=1 mg/ml; Armsbio). Spheroplasts were washed once with 5 ml sorbitol 1M, once with 5 ml 1X RE buffer (DpnII NEB buffer) and finally suspended in 3.5 ml of 1X RE buffer. The spheroplasts were treated with 3% SDS for 20 min at 65°C and the lysate was digested overnight with DpnII (CFinal=450 U/pellet; NEB) at 37°C. The digestion was centrifuged for 20 min at 18000 g, the supernatant discarded and the pellet suspended in 400 μl of water. The 5’ overhangs from DpnII digestion were filled in using dNTP 30 μM (biotin-14-dCTP, dATP, dGTP and dTTP; Invitrogen), at 37°C for 45 min. The biotinylated DNA fragments were ligated by T4 DNA ligase (CFinal=250 Weiss U/pellet; Thermo Scientific) for 4 h at 16°C. DNA purification was achieved through an overnight incubation at 65°C in presence of proteinase K 250 μg/ml and EDTA 6.2 mM, followed by a phenol/chloroform extraction on the precipitated DNA and an RNAse A DNase-free 500 μg/ml treatment. Hi-C DNA libraries were 500 bp sheared, using CovarisS220 apparatus and were used as template for the paired-end llumina sample preparation method. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase (NEB), DNA Pol I large fragment (Klenow polymerase; NEB) and T4 polynucleotide kinase (NEB). The blunt, phosphorylated ends were treated with Klenow fragment (exo minus; NEB) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters. After adapter ligation library fragments of ~500 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified biotin-labeled DNA fragments were selectively captured by Dynabeads Myone Streptavidin C1 (Invitrogen) and PCR amplified with Illumina primers for 12 cycles.
attributes.tag: GEO Accession
attributes.value: GSM2417292