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| Status |
Public on Oct 04, 2012 |
| Title |
S2-DRSC WT Late S-phase Replicate 1 extraction4_seq1 aliquote 1 |
| Sample type |
SRA |
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| Source name |
S2_WT_LateSphase_1
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2-DRSC
tissue: embryo-derived cell-line
developmental stage: late embryonic stage
Sex: Male
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| Growth protocol |
The general growth protocol for growing Drosophila cell lines in culture.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA isolation from Drosophila cell lines. RNAse-treated cellular extract is treated with ethanol in order to precipitate DNA.
Cells are pulse labeled with 50ug/ml BrdU for one hour, and washed with 1x PBS.
FACS Protocol.1. Spin cells down and resuspend in 300µl of PBS. (For large pellets increase this volume and increase the EtOH volume proportionally)2. Fix by adding 5ml of 70% EtOH Dropwise while vortexing gently, incubate at -20ºc for at least 30 minutes.3. Spin, remove EtOH and resuspend in 5ml PBS. Allow cells to rehydrate at 4ºc for at least an hour.4. Spin and resuspend the cells in 4ml of PBS with 10µg/ml RNaseA and 10µg/ml PI. Incubate at RT for at least 30min up to several hours (alternatively incubate for 10min at 37ºc)Cells were sorted into early mid and late S-phase fractions using a FACS Vantage SE (BD Biosciences) equipped with the DiVa acquisition system.
An immunoprecipitation procedure in which anti-BrdU is used to enrich for BrdU labeled DNA.
Generation of a genomic library suitable for Illumina sequencing.Generation of a genomic library suitable for Illumina sequencing. The procedure starts with random shearing of genomic DNA with sonication followed by Illumina-developed sample preparation protocol.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina_sequencing:DM:1 protocol. Load sample onto flow cell at a (usually 2-8 pM, variable) concentration and run on an Illumina HiSeq 2000 for single-end sequencing for 36 or 76 nt. Image data is then deconvoluted using the most current versions of Firecrest, Bustard, and Gerald (or equivalents). Processed data are obtained using following parameters: read length is 50 Bowtie alignment:DM:1 protocol. For paired end CNV:Using the default setting, Bowtie2 was used to align paired sequenced reads to the reference genome. Single end HiSeq RepliSeq:Bowtie was used to align sequenced reads to the reference genome, with the following parameters:--phred33-quals -p 6 -n 2 -l 20 -m 1 -k 1 -S -y --best --strata Processed data are obtained using following parameters: genome version is 5.1 Illumina sequencing WIG Generation RPKM from BAM:DM:1 protocol. For Repliseq experiments, a bin size of 10000 bp stepping every 2500 bp was used.
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| Submission date |
Oct 04, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
[email protected]
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| Phone |
416-673-8579
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| Organization name |
Ontario Institute for Cancer Research
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| Lab |
modENCODE DCC
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| Street address |
MaRS Centre, South Tower, 101 College Street, Suite 800
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
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| Platform ID |
GPL13304 |
| Series (1) |
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| Relations |
| SRA |
SRX193649 |
| BioSample |
SAMN01761682 |
| Named Annotation |
GSM1015353_dm247.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1015353_dm247.wig.gz |
86.0 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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