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Sample GSM2327657

Query DataSets for GSM2327657

Status

Public on Mar 22, 2017

Title

G1-R2

Sample type

SRA

Source name

Hi-C, WT arrested in G1, biological replicate 2

Organism

Saccharomyces cerevisiae

Characteristics

strain: W303-1a
genotype: MATa ade2-1 ura3-1 his3-11, trp1-1, can1-100 UBR1::pGAL-myc-UBR1 (HIS3), leu2-3 LEU2::pCM244 x3, cdc20-td CDC205' upstream -100 to -1 replaced with kanMX-tTA (tetR-VP16)-tetO2 - Ub -DHFRts - Myc -linker)

Treatment protocol

Cells were arrested in G1 with 10 μg/ml alpha factor until 90% of cells were unbudded (120 min). The cells were washed three times with YPR and released in YPR. After 30 min 10 μg/ml of nocodazole was added and budding was checked after 1h. After cells entered G2 (60-70 min after release), Galactose at 2% final concentration and 15 min later Doxycycline at 50μg/ml final concentration were added. 30 min after addition of Galactose, the temperature was shifted to 37 °C. Cells were grown at 37 °C for 1h. Then cells were washed three times in YPR + Galactose + Doxycycline and released in the same media. Cells were collected after 30 min for the cdc20 metaphase. For cdc45-td cells, as above except alpha factor arrested samples incubated at 37°C and 2% galactose added before releasing into the cellcycle in the same media at 37°C. Sample were then collected after 100 minutes for Hi-C analysis.

Growth protocol

Yeast cells were grown in YP + 2% Glucose at 25 °C, transferred to YP + 2% Raffinose (YPR) and grown over night to log phase.

Extracted molecule

genomic DNA

Extraction protocol

Hi-C was performed as described in (JM Belton et.al., The Conformation of Yeast Chromosome III Is Mating Type Dependent and Controlled by the Recombination Enhancer, Cell Reports, 13(9):1855-67, (2015)) with the variation that cells were fixed at 37 °C.
Hi-C: The yeast culture was crosslinked with 37% formaldehyde to a final concentration of 3% in the culture media for 20 min at 37˚C. The crosslinking was quenched by adding 2X the volume of formaldehyde used of 2.5M Glycine to the culture and incubated culture for 5 min at 25˚C. Cells were lyses by grinding in a cold mortar and pestle. The chromatin was solubilized by adding 1% w/v SDS and Incubating at 65˚C water bath for 10 min. Excess SDS was quenched using 10% v/v Triton X-100. The chromatin was digested with HindIII at 37˚C overnight in a water bath. Digested ends were filled in with biotin-14-dCTP with Klenow at 37˚C for 2 hr in a water bath. The klenow was inactivated by Incubating in a 65˚C water bath for 20 min. Crosslinked and digested fragments were ligated together in a dilute reaction inorder to favor intra-molecular ligation in a 16˚C water bath for 8 hr. Crosslinkes were reversed by incubating the ligation mixture with proteinase K in a 65˚C water bath. Ligation products were purified with phenol (pH 8.0):chloroform and precipitated with sodium acetate pH 5.2 and ethanol. The precipitated DNA was resuspended in 1X TE and then the DNA was re-extracted with phenol (pH 8.0):chloroform and precipitated with sodium acetate pH 5.2 and ethanol. The DNA pellet was dried thoroughly and excess salt was washed out of the solution using an amicon 30kDa spin column using 1X TE. Then the sample was concentrated using a 30kDa amicon column. RNA was degrade using DNase free RNase A at 37˚C for 1 hr. The Hi-C library was quantified on a 0.8% agarose gel in 0.5X TBE by comparing it to known concentration standards. The Hi-C efficiency was accessed by first PCR amplifing a neighboring interaction in the Hi-C library. This amplicon was split and the aliquots were digested with either HindIII, NheI, or HindIII and NheI at 37˚C overnight. The digested products were quantified on a gel and the percent Hi-C efficiency was calculated as the percent digested with NheI divided by the percent digested in the NheI and HindIII combined reaction. Biotin was removed from un-ligated ends using T4 DNA polymerase at 20˚C for 4hrs and then at 75˚C for 20 min to inactivate the enzyme. The DNA was sheared to 50-700bp using the Covaris S2 sonicator. The DNA ends were repaired using T4 DNA Polymerase, T4 Polynucleotide Kinase, and Klenow fragment of DNA Polymerase I at 20˚C for 30 min. The DNA was purified with one Qiagen MinElute Column. A dATP was added to the 3’ end of the molecules by using Klenow Fragment (exo-) at 37˚C for 30 min and then at 65˚C for 20 min to inactivate the enzyme.
Hi-C library construction was performed as described in (JM Belton et.al., The Conformation of Yeast Chromosome III Is Mating Type Dependent and Controlled by the Recombination Enhancer, Cell Reports, 13(9):1855-67, (2015)). The end repaired and A-tailed library was fractionated to 100-300bp using 0.9x and then 1.1x ampure XP extractions. For each 1.0 ng of biotinylated ligation products, 1.0 µl of Dynabeads MyOne Strepavidin C1 beads were used to enrich the library. Illumina PE adapters were ligated to the library using NEB Quick Ligase at room temperature for 15 min. The library was amplified using Illumina primers PE1.0 and PE2.0 for as few cycles as possible. Cycle number was determined by a titration experiment that revealed which cycle would produce enough library for sequencing but didn’t produce higher molecular weight artifacts. The library was purified once more with 1.8x Ampure XP to remove primers and primer dimers. The final library was quantified on a bioanalyzer. The libraries were sequenced on either the GAII or HiSeq platforms

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

G1-R2

Data processing

Library strategy: Hi-C
Mapping and filtering contacts: We mapped sequenced read pairs to W303 yeast genome using Bowtie 2.1.0 and the previously described method of iterative mapping (Imakaev, M. et al. Iterative correction of Hi-C data reveals hallmarks of chromosome organization. Nat. Methods 9, 999-1003, doi:10.1038/nmeth.2148 (2012)). To generate contact lists, we assigned each mapped side to a HindIII fragment and removed the contacts with both sides assigned to the same HindIII fragment, reads with one unmapped side as well as PCR duplicates, i.e. identical contact pairs.
Aggregating and normalizing contact maps: To generate Hi-C contact maps, we aggregated filtered contact lists into 10kb genomic bins. We removed the contacts within the first two diagonals of the maps as they are contaminated by uninformative Hi-C artifacts, unligated and self-ligated DNA fragments. Finally, we iteratively corrected the resulting maps to equalize genomic coverage.
Genome_build: w303
Supplementary_files_format_and_content: matrices.tar.gz - files contain sparse tables with Hi-C contact maps at 10kb resolution.

Submission date

Sep 23, 2016

Last update date

May 15, 2019

Contact name

Anton Goloborodko

E-mail(s)

goloborodko.anton@gmail.com

Organization name

MIT

Department

Physics