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Sample GSM4262942

Query DataSets for GSM4262942

Status

Public on Jan 22, 2020

Title

HomoSap.RepTiming.25hr.Replicate3

Sample type

SRA

Source name

in vitro cell culture

Organism

Homo sapiens

Characteristics

cell line: MDA-MB-231

Treatment protocol

For G2/M arrest, 2.0x10e6 cells per p15 tissue culture plate were grown for 48 hours, followed by treatment with 30uM BrdU for 4 hours prior to addition of nocodazole to 12.5ng/ml for an additional 12 hours. For release, non-adhering G2/M phase arrested cells were tapped and rinsed off the plate, washed twice with media via centrifugation at 1000 rpm for 3 minutes and resuspened in 10ml fresh complete DMEM, then plated on new 10cm plates in 10ml media containing 30uM BrdU. Samples were collected at 0, 10, 15 and 25 hours after release for DNA and flow cytometry analysis.

Growth protocol

MDA-MB-231 cells were maintained in DMEM with 10% fetal bovine serum, 1% penicillin/streptomycin, and L-glutamine at 37 degrees Celsius with 5% CO2. 7.5 x 105 cells were seeded on 10 cm plates in 10 ml complete DMEM and allowed to adhere and grow for 48 hours prior to treatment.

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

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Description

Human replication timing samples
HomoSap.RepTiming.25hr.merged.bedgraph

Data processing

Human fastq files were aligned to HG38 using bowtie2
The resulting sam files were turned into bams and duplicate reads were removed using samtools
Bam files were turned into bedgraphs using the bedtools2.29.2
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 1kb and smoothed using a 100kb sliding window
To make the final processed files, replicates were merged and averaged to make a single bedgraph
Genome_build: Hg38
Supplementary_files_format_and_content: Bedgraph normalized by RPM and mitochondria content, merged across replicates
Library strategy: Rerep-seq

Submission date

Jan 13, 2020

Last update date

Jan 22, 2020

Contact name

Joshua C Black

E-mail(s)

blacklabucd@gmail.com

Organization name

University of Colorado

Department

Pharmacology