GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM498668 Query DataSets for GSM498668
Status Public on Jan 21, 2010
Title cnv_S2-DRSC_1 extraction1_seq1
Sample type SRA
Source name cnv_S2-DRSC_1 extraction1_seq1 channel_1
Organism Drosophila melanogaster
Characteristics cell line: S2-DRSC
tissue: embryo-derived cell-line
developmental stage: late embryonic stage
sex: Male
Growth protocol Cell growth protocol;
Grow 10ml of cells in 100mm dishes. For Kc and S2 cells use: Schneider's medium (invitrogen 11720-067) 10% ModEncode serum 1% Penicillin/Streptomycin/Glutamine (PSG) (Invitrogen 10378-016). For ML-DmBG3-c2 cells use: Schneider's medium 10% ModEncode serum 1% PSG 10ug/ml human insulin (sigma I9278). Cells are grown at 25C. Cells are happiest between 1.5X10^6 and 2.0X10^6
Extracted molecule genomic DNA
Extraction protocol DNA isolation protocol;
1. Grow 10ml of cells to 1.5 x 106 in 100mm dishes
2. Harvest cells into 15ml conical tubes
3. Pellet cells at 1000g for 5 minutes
4. Wash cells once in cold 1X PBS
5. Resuspend pellet in 0.7ml 10mM Tris pH 9.5
6. Add 0.7ml NDS. Invert to mix
7. Add 0.1ml 20mg/ml Proteinase K (Amresco 0706-1G). Invert to mix
8. Inc. 37C, 2 hours
9. Add 1.5ml 1X TE. Invert to mix
10. Add 3.0ml phenol/chloroform. Invert tubes over 5 minutes
11. Centrifuge 3500 rpm for 10-12 minutes
12. Remove top layer with cut p1000 tips to a new 15ml tube
13. Repeat steps 10-12
14. Add 2.5 volumes of room temp. 100% Ethanol. Invert
15. Inc. at room temp overnight
16. Centrifuge tubes 3500-3700rpm for 10-12 minutes
17. Wash pellet with 70% Ethanol and transfer pellet to a 1.5ml tube
18. Centrifuge full speed for 5 minutes
19. Air dry pellet
20. Resusp. pellet in 250ul 1X TE
21. Add 5ul 10mg/ml RNaseA (Amresco 0675-250mg). Inc. 37C for 1h
22. Add 1/10 volume 3M NaOAc. Vortex
23. Add 2X volume cold 100% Ethanol. Vortex
24. Inc. -80C 20-30 minutes
25. Centrifuge at 4C for 15 minutes
26. Wash with 70% Ethanol
27. Air dry pellet. Resusp. in 250ul 1X TE
28. Spec DNA Shearing DNA
29. Shear DNA 3X 10 seconds with 1minute on ice in between Optional [for BrdU experiments]
30. Run 5ug on 1% agarose gel
31. Set up Southern overnight
32. Check for BrdU incorporation by western. anti-BrdU antibody (BD BioSciences 555627 ) is 1:1000 dilution in milk/TBS-T

Genomic DNA library protocol;
1. Genomic DNA was fragmented to less than 800bps by sonication
2. the library was prepared as described in the Illumina provided protocol "Preparing samples for sequencing genomic DNA" (part # 11251892 Rev. A) with the exception of an additional gel extraction (size select for 150-200bps) after the "enrich the adapter-modified DNA fragments by PCR" step.
3. Samples were submitted to the Duke University IGSP sequencing facility.
Library strategy WGS
Library source genomic
Library selection RANDOM
Instrument model Illumina Genome Analyzer
Data processing Illumina_seq_maq_processing: DM:1 protocol;
Get Illumina FASTQ files back from Duke IGSP Sequencing Facility. Their steps, briefly:
* Image analysis (SCS real time analysis software / Firecrest software)
* Base calling (
* Sequence analysis ( -> results in the FASTQ file that gets sent to us
* Using maq command-line tool v. 0.7.1:
* Convert Illumina FASTQ file to Sanger-style FASTQ format: maq sol2sanger $workingdir/$solfastqfilename $workingdir/$solfastqfilename.sangerfastq
* Convert Sanger FASTQ file to Binary FASTQ format: maq fastq2bfq $workingdir/$solfastqfilename.sangerfastq $workingdir/$solfastqfilename.sangerbfq
* Convert Drosophila 5.1 FASTA file to Binary FASTA format: maq fasta2bfa $workingdir/$referencefastafile $workingdir/$referencefastafile.bfa
* Map reads back to Drosophila genome allowing 3 mismatches: maq map -n 3 $workingdir/$ $workingdir/$referencefastafile.bfa $workingdir/$solfastqfilename.sangerbfq
* Create a mapview file to import into the database: maq mapview $workingdir/$ > $workingdir/$destination_table.mapview

Processed data are obtained using following parameters:
genome version is 5.1
read length is 36

CNV_seq_analysis:DM:1 protocol
* Read locations are shifted:
* Positive reads are shifted downstream by half of the estimated sonication size
* Negative reads are shifted upstream by half of the estimated sonication size
* Reads with a map score (Phred) >= 35 (leading to maximum probability that a read is misaligned of ~0.03%) are then binned based on their location, with non-overlapping bins of size 1000bp covering the genome
* The total number of reads in each bin are summed across replicates
Submission date Jan 20, 2010
Last update date May 15, 2019
Contact name DCC modENCODE
Phone 416-673-8579
Organization name Ontario Institute for Cancer Research
Street address MaRS Centre, South Tower, 101 College Street, Suite 800
City Toronto
State/province Ontario
ZIP/Postal code M5G 0A3
Country Canada
Platform ID GPL9058
Series (1)
GSE19956 CNV S2-DRSC Cells
SRA SRX015838
BioSample SAMN00007547

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap