|
| Status |
Public on Nov 30, 2012 |
| Title |
Shep (alternate antibody) |
| Sample type |
SRA |
| |
|
| Source name |
BG3 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
antibody: Shep, rabbit
|
| Growth protocol |
BG3-c2 cells were grown in S2 medium (Sigma) supplemented with 10% fetal calf serum and 10 µg/mL insulin. Cells were maintained in monolayer at 25˚C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed in 1% formaldehyde added directly to cells in culture medium for 10 min at RT with gentle agitation; formaldehyde was quenched by addition of glycine to 0.125 M with gentle agitation for 5 min at RT. 5 x 106 to 107 cells were used per IP. Cells were pelleted at 400 rcf and washed twice in ice cold PBS. Cells were resuspended in 1 mL ice cold cell lysis buffer (5 mM PIPES, pH 8, 85 mM KCl, 0.5% NP-40) supplemented with protease inhibitors, and nuclei were released by Dounce homogenization with the B pestle and pelleted by centrifugation at 9190 rcf for 5 min at 4˚C. Nuclei and chromatin were further processed as described (Moshkovich and Lei, 2010).
Libraries were constructed according to the Illumina standard ChIP-seq protocol (Part # 11257047 Rev. A) with TruSeq adapters and sequenced on an Illumina HiSeq multiplexed in a single lane.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
ChIP-seq of Shep, second antibody
|
| Data processing |
Aligned to dm3 using Bowtie 0.12.7, parameters –best –strata –sam -m1 -n2 –tryhard -k1
Removed duplicates using Picard 1.49 MarkDuplicates
Removed reads falling in RepeatMasked regions (bedtools intersect, v2.17.2)
Peak calling using spp using default parameters except srange=c(50,200) for get.binding.characteristics(); FDR=0.01 for find.binding.positions(). Final broad peak regions were merged with bedtools merge (v2.17.2)
BigWigs created using bedtools genomecov (v2.17.2) with the -scale parameter set to 1/(mapped reads / 1e6) to convert BAM to scaled bedGraphs, then UCSC's bedGraphToBigWig to convert to bigWig format
Genome_build: dm3, excluding chrUextra
Supplementary_files_format_and_content: BigWig: Conversion of BAM to bigWig format. BED: Called peaks from spp.
|
| |
|
| Submission date |
Sep 12, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Ryan Dale |
| E-mail(s) |
dalerr@nih.gov
|
| Organization name |
National Institutes of Health
|
| Department |
National Institute of Child Health and Human Development
|
| Lab |
Bioinformatics and Scientific Programming Core
|
| Street address |
Rm 10D39, 10 Center Drive
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL13304 |
| Series (1) |
| GSE40797 |
Tissue-specific regulation of chromatin insulator function |
|
| Relations |
| SRA |
SRX186111 |
| BioSample |
SAMN01166076 |
| Named Annotation |
GSM1001883_shep-2.bigwig |
