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| Status |
Public on Apr 16, 2010 |
| Title |
S2 RNA |
| Sample type |
SRA |
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| Source name |
S2 cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2, ATCC, CLR-1963, Lot#5054622
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| Growth protocol |
The Drosophila S2 cells were cultured following the manufacturer’s protocol. Exponentially growing S2 cells were harvested and resuspended in digestion buffer (50mM Tris-HCl, pH7.6, 1mM CCl2, 0.2% Triton X-100, 5mM butyrate, 1x proteinase inhibitor cocktail and 0.5mM PMSF).
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| Extracted molecule |
total RNA |
| Extraction protocol |
For ~20 µg total RNA from S2 cells, we performed two rounds of mRNA isolation using Dynabeads mRNA purification kit (Invitrogen, #610-06), according to the manufacturer’s instructions. The final mRNAs were eluted in 13.5 µl 10 mM Tris-HCl (pH 7.5) and immediately used to generate the first strand cDNA, using 4 µl random hexamers (ABI, #N8080127) and SuperScript II Reverse Transcription Kit (Invitrogen, #18064-014) in a 30 µl final volume, following the manufacturer’s instructions. The second strand cDNA was generated with the following recipe: 10 µl 5´ second strand buffer (500mM Tris-HCl pH7.8, 50mM MgCl2, 10mM DTT), 30nmol dNTPs (Invitrogen, #18427-013), 2 Units of RNase H (Invitrogen, #18021-014) and 50 Units of DNA Pol I (Invitrogen, #18010-025). The entire reaction mix was incubated at 16°C for 2.5 hours. The double-stranded DNA (dsDNA) was purified with QIAquick PCR purification kit (Qiagen, #28106) and the concentration was quantified by the Qubit fluorometer (Invitrogen).
To generate sequencing libraries, about 300 ng dsDNA of each sample was fragmented by sonication using Bioruptor (Diagenode, UCD-200-TM-EX) under the following conditions: medium power output for 30 min in ice water. The resulting DNA fragments were analyzed by agarose gel to verify they are within ~100-300-bp size range. Sequencing libraries were prepared as the follows: end-repair (DNA end-repair kit from Epicenter, #ER0720); A-tailing (300 ng dsDNA, 5 µl Thermo buffer, 10 nmol dATP, 15 Units of Taq polymerase, at 70oC for 30min); Solexa adaptor ligation (300 ng dsDNA, 4 µl DNA Ligase buffer, 1 ul Solexa adaptor mix, 3 ul DNA Ligase, at 70oC overnight.); PCR (98°C 10 sec, 65°C 30 sec, 72°C 30 sec for 16 cycles; then additional 72°C for 5 min) amplification with adaptor primers and size selection (200-400bp). Then the library dsDNA for S2 cells was used on an Illumina Genome Analyzer (GA) II at a concentration of 10ng per lane
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
RNA sequencing
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| Data processing |
The quality-filtered 36 bp short sequence reads were aligned to the reference sequence consisting of dm3 Drosophila melanogaster genome sequences using ELAND (Efficient Local Alignment of Nucleotide Data) software, allowing up to two mismatches with the reference sequence for the first 32bp. To calculate a single number to represent expression level of a gene, all exon regions belonging to a gene are merged and the total number of non-redundant unique reads in the resulting merged exonic region is counted. The resulting number of transcript abundance for the genes was computed as sequencing reads/per kilobase merged exonic region/per million mapped reads (RPKM value provided in GSM480160_GA0840_Drosophila_S2_RNAseq.xls file).
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| Submission date |
Dec 04, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Dustin E Schones |
| E-mail(s) |
[email protected]
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| Organization name |
City of Hope
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| Street address |
1500 E Duarte Rd.
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| City |
Duarte |
| State/province |
CA |
| ZIP/Postal code |
91010 |
| Country |
USA |
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| Platform ID |
GPL9061 |
| Series (1) |
| GSE19325 |
Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis |
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| Relations |
| Reanalyzed by |
GSM3274595 |
| SRA |
SRX017853 |
| BioSample |
SAMN00010228 |
| Named Annotation |
GSM480160_GA0840_Drosophila_S2_RNAseq.bed.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM480160_GA0840_Drosophila_S2_RNAseq.bed.gz |
42.5 Mb |
(ftp)(http) |
BED |
| GSM480160_GA0840_Drosophila_S2_RNAseq.xls.gz |
833.0 Kb |
(ftp)(http) |
XLS |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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