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| Status |
Public on Jan 22, 2020 |
| Title |
SacCer.cdkByPass.Galacose.Replicate1 |
| Sample type |
SRA |
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| Source name |
in vitro cell culture
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: MATa ade2-1 ura3-1 his3-11,15:: HIS3 GPD-HSV-TK ADH1-hENT1 BrdU-Inc trp1-1 leu2-3,112 can1-100 {delta}bar1::hisG CDC45 H22Y ura3-1::GAL-sid2-11D-MHT (URA3) leu2-3,112::GAL-DBF4-MHT (LEU2)
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| Treatment protocol |
Cells were grown in YP 2% raffinose to an OD600 of 0.5, arrested in G1 with α-factor (100 ng/ml) for 2 hours. 0.1mM BrdU was added and the culture was split into two, with 2% galactose added to one for induction of cdc45 H22Y and sld2-11D, while the other was maintained in 2% raffinose. Samples were collected at 0 and 5 hours for flow cytometry and DNA extraction
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| Growth protocol |
Yeast strains were grown according to standard procedures in YPD (yeast extract, peptone, and 2% dextrose) at 30ºC
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA extraction: As RerepSeq selectively fragments rereplicated DNA and enriches those sequences by size selection, it is imperative that the genomic input DNA is of high molecular weight. For yeast samples: cells were pelleted by centrifugation at 3000 rpm for 3 minutes and resuspended in SCE (1M sorbitol, 100 mM sodium citrate, 10 mM EDTA pH 8.0); fresh 0.125% (v/v) β-mercaptoethanol and 10 U/mL zymolyase was added and incubated for 30-60 minutes at 37ºC to digest cell walls; human cells or yeast spheroplasts were pelleted by centrifugation at 3000 rpm for 3 minutes and resuspended in 500uL RIPA buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM PMSF) with RNase A, 0.2 mg/mL and incubated at 37ºC for 1 hour. 25uL 20% SDS and 10µl of 20 mg/ml proteinase K was then added and allowed in incubate at 55ºC for 2 hours. DNA was extracted twice using Phenol:Chloroform:Isoamyl alcohol (25:24:1, v/v) and precipitated using isopropanol. DNA was resuspended and stored in nuclease free water. RerepSeq Digestion: In an 8 strip 200 µl PCR tube 1 – 5 µg (depending on the experiment) high molecular weight genomic DNA was mixed with 2.5uL 10X Hoechst 33258 (0.1mg/ml) and 2.5uL 10X NEB Buffer 4 (50 mM Potassium Acetate, 20 mM Tris-acetate , 10 mM Magnesium Acetate, 1 mM DTT pH 7.9@25°C) to a final volume of 24uL; open tubes placed upright in PCR tube rack, covered with glass plate (3" x 3" glass plate from VWR Vertical Gel Electrophoresis Systems), exposed to 7.5 minutes of glass plate filtered (UVA only) from a Stratalinker. Following UVA treatment, samples were digested with 0.5uL UDG (5 units of Uracil-DNA Glycosylase), 0.5uL APE1 (10 units of human apurinic/apyrimidinic endonuclease 1) for 2 hours at 37C. Digested DNA was repaired with NEB’s FFPE DNA Repair Mix for 30 minutes then separated on 0.8% agarose gel for 15 minutes. Fragmented DNA ranging from 0.1Kb to 3Kb was gel extracted with Wizard® SV Gel and PCR Clean-Up System (Promega) and the purified DNA was resuspended in 50uL of water for subsequent qPCR and sequencing analysis.
Illumina Nextera DNA Flex Library Prep Kit
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Yeast CDK bypass
SacCer.cdkByPass.Galacose.merged.bedgraph
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| Data processing |
Yeast fastq files were aligned to SacCer3 using bowtie3
The resulting sam files were turned into bams and duplicate reads were removed using samtools
Bam files were turned into bedgraphs using the bedtools2.29.3
If samples were sequenced for additional depth, the bam files were merged at this point
Bedgraphs were read count normalized using an RPM calculation
RPM normalized bedgraphs were then scaled to mitochondria read percentages
The fully normalized bedgraphs were binned to 100bp and smoothed using a 10kb sliding window
To make the final processed files, replicates were merged and averaged to make a single bedgraph
Genome_build: SacCer3
Supplementary_files_format_and_content: Bedgraph normalized by RPM and mitochondria content, merged across replicates
Library strategy: Rerep-seq
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| Submission date |
Jan 13, 2020 |
| Last update date |
Jan 22, 2020 |
| Contact name |
Joshua C Black |
| E-mail(s) |
blacklabucd@gmail.com
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| Organization name |
University of Colorado
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| Department |
Pharmacology
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| Street address |
12801 East 17th Ave
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| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
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| Platform ID |
GPL21656 |
| Series (1) |
| GSE143572 |
Isolation and analysis of rereplicated DNA by Rerep-seq |
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| Relations |
| BioSample |
SAMN13838299 |
| SRA |
SRX7549523 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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