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Sample GSM4946279 Query DataSets for GSM4946279
Status Public on Jun 15, 2022
Title WT, HU [KJ20]
Sample type SRA
 
Source name WT
Organism Saccharomyces cerevisiae
Characteristics strain: CB67
treatment: HU-arrest
Treatment protocol For transcription shut-off and degradation of Scc2 and Wpl1 in a synchronized S-phase, auxin and doxycycline were first added to G1-arrested cells for 30 minutes, and then for an additional hour in the HU-containing release medium at the same final concentrations as above. For transcription inhibition, thiolutin (Abcam, ab143556) was added to cell cultures for the final concentration of 20 µg ml-1 for 30 minutes.
Growth protocol Cells were cultured in YEP medium (1 % yeast extract, 2 % peptone, 40 μg ml-1 adenine) supplemented with 2 % glucose (YEPD). For arrest in G2/M, benomyl (Sigma, 381586)-containing YEPD medium was added to cells growing logarithmically for a final concentration of 80 µg ml-1. Cell cultures were then incubated for 90 minutes at 30°C achieving complete G2/M-arrest. For synchronization in S-phase, 3 μg ml-1 α-factor mating pheromone (Sigma, custom peptide WHWLQLKPGQPMY) was added every hour (a total of three additions) to cells growing logarithmically at 25°C. Upon complete G1-arrest, cells were released into medium containing 0.2 M hydroxyurea (HU) (Sigma, H8627), and S-phase was allowed to progress at 25°C. For transcription shut-off and degradation of Scc2 and Wpl1 in G2/M-arrest, auxin (3-indoleacetic acid, Sigma, I2886) and doxycycline (Sigma, D9891) were added for 1 hour at the final concentration of 1 mM and 5 μg ml-1, respectively.
Extracted molecule genomic DNA
Extraction protocol Our Hi-C protocol was adapted for S. cerevisiae from ref. (Rao, S. S. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665-1680, doi:10.1016/j.cell.2014.11.021 (2014)). Fifty ml of cell culture (at an OD of 1.0) was fixed with 3 % formaldehyde for 20 minutes at 30°C, before the reaction was quenched by adding glycine to 0.125 M final concentration for 5 minutes at room temperature. Cells were washed once with 1 x PBS, before being resuspended in 5 ml pre-spheroplasting buffer (100mM PIPES (pH 9.4), 10mM DTT). The cells were incubated 5 minutes at room temperature and then pelleted (1500 g for 5 minutes at room temperature), before being resuspended in 5 ml spheroplasting buffer (50mM KH2PO4/K2HPO4 (pH 7.5), 1 M Sorbitol, 10 mM DTT). Twenty-five μl of 10 mg ml-1 100T Zymolyase (Nacalai Tesque, 07665-55) was added and cells were incubated for 15 minutes at 30°C. The spheroplasts were pelleted (500 g for 5 minutes at 4°C) and washed twice with ice-cold spheroplasting buffer (containing only 1 mM DTT). The spheroplasts were then resuspended in 250 μl of ice-cold Hi-C lysis buffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.2 % Igepal CA630) supplemented with 50 μl 6 x Complete (Roche) and 3 μl protease inhibitor (Sigma, P8215), and incubated on ice for 15 minutes. The spheroplasts were pelleted (1500 g for 5 minutes at 4°C) and washed twice with 500 μl of ice-cold Hi-C lysis buffer. After the last wash step the supernatant was discarded and the spheroplasts were incubated for 6 minutes at 62°C. Thereafter sodium dodecyl sulphate (SDS) was added to a final concentration of 0.2 % and the reaction was immediately and thoroughly mixed by inversion, and thereafter incubated at 62°C for 10 minutes. After addition of 80 μl H2O, 25 μl 10 % Triton X-100 was added to quench the SDS. The reaction was mixed by inversion and incubated at 37°C for 15 minutes. Thereafter, 28 μl 10 x NEB DpnII buffer and 500 units of DpnII (NEB, R0543M) was added, and the chromatin was digested overnight at 37°C. At the end of the incubation, the reaction was supplemented with 250 units of DpnII and incubated for 1 hour at 37°C. Then, the restriction enzyme was inactivated at 62°C for 20 minutes. The presence of intact and individual DNA masses throughout the spheroplasting, digestion and ligation steps was confirmed by DAPI (4′,6-diamidino-2-phenylindole)-staining and microscopy.
Marking and repairing DNA ends, proximity ligation, crosslink reversal, DNA shearing, size selection, biotin pull-down, preparation for Illumina sequencing, final amplification (15 cycles) and purification was performed as in ref. (Rao, S. S. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665-1680, doi:10.1016/j.cell.2014.11.021 (2014)).
 
Library strategy Hi-C
Library source genomic
Library selection other
Instrument model HiSeq X Ten
 
Description KJ20
Data processing Sequenced paired-end 150-bp reads were mapped to the yeast genome using Juicer with default parameters.
Genome_build: Saccharomyces Genome Database (SGD), identical with sacCer3.
Supplementary_files_format_and_content: hic files were generated by juicertools version 1.9.9 pre with "-r 1000" option. The total hic contact number of Hi-C samples was normalized.
 
Submission date Nov 25, 2020
Last update date Jun 15, 2022
Contact name Ryuichiro Nakato
E-mail(s) rnakato@iqb.u-tokyo.ac.jp
Phone +81-3-5841-1471
Organization name The University of Tokyo
Department Institute for Quantitative Biosciences
Lab Laboratory of Computational Genomics
Street address 1-1-1 Yayoi
City Bunkyo-ku
State/province Tokyo
ZIP/Postal code 113-0032
Country Japan
 
Platform ID GPL26171
Series (2)
GSE162193 Cohesin-dependent chromosome loop extrusion is limited by transcription and stalled replication forks [Hi-C]
GSE162194 Cohesin-dependent chromosome loop extrusion is limited by transcription and stalled replication forks
Relations
BioSample SAMN16924747
SRA SRX9588257

Supplementary file Size Download File type/resource
GSM4946279_KJ20.hic 20.2 Mb (ftp)(http) HIC
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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