|
| 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 ( Bustard.py)
* Sequence analysis (GERALD.py) -> 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 $workingdir/$destination_table.map $workingdir/$referencefastafile.bfa $workingdir/$solfastqfilename.sangerbfq
* Create a mapview file to import into the database: maq mapview $workingdir/$destination_table.map > $workingdir/$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 |
| E-mail(s) |
help@modencode.org
|
| Phone |
416-673-8579
|
| Organization name |
Ontario Institute for Cancer Research
|
| Lab |
modENCODE DCC
|
| 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) |
|
| Relations |
| SRA |
SRX015838 |
| BioSample |
SAMN00007547 |
