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| Status |
Public on Dec 07, 2011 |
| Title |
roX2 CHART RNase eluted rep 2 |
| Sample type |
SRA |
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| Source name |
S2 cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: MSL3-TAP
cell type: S2 cells
sample type: roX2 CHART
elution strategy: Rnase-eluted
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| Treatment protocol |
Extract (250 µL, 8x107 cell equiv.) was adjusted to hybridization conditions (20mM HEPES pH7.5, 817mM NaCl, 1.9M Urea, 0.4% SDS, 5.7mM EDTA, 0.3mM EGTA, 0.03%NaDeoxycholate, 5XDenhardt’s) and pre-cleared with ultralink-streptavidin resin. For biotin eluted CHART: Capture oligonucleotides (800 nM ea. R2.1-3) were added and hybridized (55°C 20min; 37°C 10min; 45°C 60min; 37°C for 30min). The bound material was captured using streptavidin beads rinsed and eluted with biotin. For RNase H eluted CHART: Capture oligonucleotides 1 µM nM ea. R2.1b-3b) were added and incubated overnight at room temperature. The biotin-bound material was captured using streptavidin beads, rinsed and eluted with RNase H.
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| Growth protocol |
Drosophila S2 cells stably transfected with a plasmid expressing MSL3-TAP (Alekseyenko et al. High-resolution ChIP-chip analysis reveals that the Drosophila MSL complex selectively identifies active genes on the male X chromosome. Genes & Development (2006) vol. 20 (7) pp. 848-57) were grown in shaker flasks in serum-free CCM3 medium (Hyclone).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Libraries were constructed using a modified version of the Illumina PE ChIP-seq protocol.
Average insert size: 233 bp
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
CHART-seq study
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| Data processing |
DNA fragments were mapped to the Drosophila genome (dm3, Bowtie aligner, see: Langmead B, Trapnell C, Pop M, & Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25), recording positions of uniquely-mappable reads. The enrichment of biotin-CHART signal was determined relative to the sense-oligo controls and RNase H-eluted CHART signal was determined relative to input. Conservative enrichment profiles were determined using SPP package (Kharchenko PV, Tolstorukov MY, & Park PJ (2008) Design and analysis of ChIP-seq experiments for DNA-binding proteins. Nat Biotechnol 26(12):1351-1359), (lower bound of enrichment was determined based on a Poisson model, with a confidence interval of p = 0.001). Positions of top CHART sites were determined as peaks of the conservative enrichment profiles (with minimum separation of 3 kb).
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| Submission date |
Nov 16, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Mark L Borowsky |
| E-mail(s) |
borowsky@molbio.mgh.harvard.edu
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| Organization name |
MGH
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| Department |
Molecular Biology
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| Street address |
185 Cambridge Street
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
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| Platform ID |
GPL11203 |
| Series (1) |
| GSE28180 |
The Genomic Binding Sites of an Endogenous Non-Coding RNA |
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| Relations |
| SRA |
SRX106073 |
| BioSample |
SAMN00760012 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM833476_roX2.2b.bed.gz |
60.7 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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