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| Status |
Public on Jul 29, 2014 |
| Title |
kc_input_3_hiseq |
| Sample type |
SRA |
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| Source name |
Kc167 cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: Kc167 cells
machine: HiSeq 2000
antibody: input
quality score: PHRED64
biological replicate: 3
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| Extracted molecule |
total RNA |
| Extraction protocol |
BG3 and Kc nuclear extracts were prepared from 3.0 x 108 to 6.3 x 109 cells per IP essentially as described in PMID: 16862159 with the following modifications: cell extracts were immediately cleared by centrifugation after dounce homogenization and purified nuclei were lysed by dounce homogenization with the B pestle in HBSMT (50 mM HEPES, pH 6.7; 150 mM NaCl; 5 mM KCl; 2.5 mM MgCl2; 0.3% Triton X-100) supplemented with 1 mM PMSF, Complete EDTA-free protease inhibitor (Roche), and 1 U/uL RNasin (Promega).
Nuclear extracts were incubated rotating for 1 h at 4˚C with Protein A conjugated sepharose (GE Healthcare) prebound to preimmune serum. Precleared extracts were then transferred to Protein A sepharose prebound to Shep or Su(Hw) antisera [PMID: 23209434] for 1 h at 4˚C, rotating. Beads and bound complexes were washed in HBSMT three times and HBSM twice. RNA was isolated directly from Protein A beads by acid:phenol extraction, and EtOH precipitated with NaOAc. RNA was DNase I treated, poly(A)+ RNA was selected using a MicroPoly(A)Purist column (Ambion), and 100 ng was cloned using the standard Illumina mRNA-seq v2.2 cloning protocol. Libraries were sequenced using commercial sequencing primers. Highly similar profiles were obtained using two independent Shep antibodies; therefore, the antibody displaying the highest signal to noise ratio was utilized for subsequent analyses.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
nuclear RNA
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| Data processing |
trim adapters with cutadapt v1.2.1; --quality-cutoff=20 –minimum-length=36 –quality-base was adjusted as appropriate for each library
map to UCSC's dm3 using TopHat2/Bowtie2, using –phred64-quals where appropriate
count reads with htseq-count, using annotations from FlyBase r5.54
sum read counts for technical replicates (same biological sample on different sequencing machine)
RIP-enrichment calculated with DESeq, with method=”per-condition” and fitType=”local”
Genome_build: UCSC dm3
Supplementary_files_format_and_content: Tab-delimited text files of read counts from htseq-count, one file per biological replicate. Column 1 is FlyBase r5.54 accession; column 2 is sum of read counts across technical replicates.
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| Submission date |
Mar 13, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Ryan Dale |
| E-mail(s) |
[email protected]
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| Organization name |
National Institutes of Health
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| Department |
National Institute of Child Health and Human Development
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| Lab |
Bioinformatics and Scientific Programming Core
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| Street address |
Rm 10D39, 10 Center Drive
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE55894 |
Su(Hw) and Shep RIP-seq in Kc and BG3 cell lines |
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| Relations |
| BioSample |
SAMN02688980 |
| SRA |
SRX488784 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1348084_kc_input_3.counts.txt.gz |
60.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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