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| Status |
Public on Dec 01, 2011 |
| Title |
3'end-seq for sorted sample |
| Sample type |
SRA |
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| Source name |
sorted nuclei
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: JM149
tissue: intenstine
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| Growth protocol |
Nuclei preparations for microscopy analysis were prepared at small scale (two to four 9 cm NGM plates) and those for flow cytometry experiments at large scale. For large scale assays, mixed stage 150 ml liquid cultures, grown at 25C, were harvested by three washes in M9 buffer (22 mM KH2PO4, 33.71 mM Na2HPO4, 85.56 mM NaCl, 1 mM MgSO4) and incubated for 30-45 min at 25C in M9 buffer for elimination of intestinal bacteria. The worm solution was subsequently passed through a 70 mm cell strainer (BD Biosciences) and the filtrate collected in 10 ml NBP (10 mM Hepes pH 7.6, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 0.25 mM sucrose) per 250 ml worm slurry (estimated before filtration) for quick buffer exchange and enrichment of adults.
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
The RNA was extracted using Trizol from Invitrogen, following the manufacturer's instructions but adding 20 ug glycogen to each Eppendorf tube as an RNA carrier.
RNA isolated from input or sorted nuclei (about 65 ng) was amplified using the MessageAmp II aRNA amplification kit (Applied Biosystems) to create antisense RNA (aRNA). Briefly, the RNA was reverse transcribed using a primer (R1) containing the T7 promoter sequence and 24 Ts. After 2nd strand synthesis, double-stranded cDNAs were in vitro transcribed by T7 RNA polymerase to give rise to aRNAs. About 30 ng of aRNA per sample was used for ligation with a 3' adapter (Bioo Scientific), which is 5' adenylated and 3' blocked. Ligation was carried out at 22oC for 1 hr using truncated RNA ligase2 (Bioo Scientific). Ligated RNA was then reverse transcribed using Superscript II reverse transcriptase (Invitrogen), followed by 12 cycles of PCR amplification using a three primer mix (P1, P2 and P3). Primers P1 and P2 were used at the ratio of 1:10. P2 and P3 contained sequences for cluster generation on the Illumina flow cell. P3 also contained an index sequence for multiplex sequencing. The amplified PCR product was run in an 8% acrylamide gel, and the product corresponding to insert size of ~50-60 nt were excised from the gel and eluted overnight. Eluted DNA was purified by ethanol precipitation and was checked in an Agilent Bioanalyzer using the high sensitivity DNA kit (Agilent technologies). cDNA were then sequenced on an Illumina GA IIx.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Sequencing reads (72 nt) were first trimmed to 50 nt and then aligned to the WS190 genome using TopHat (Trapnell et al. 2009) allowing 2 mismatches. Only reads uniquely aligned to the genome were used for subsequent analyses.
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| Submission date |
Sep 15, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Zhe Ji |
| E-mail(s) |
zhe.ji@northwestern.edu
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| Organization name |
Northwestern University
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| Street address |
303 E Superior St, Lurie 6-107
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| City |
Chicago |
| State/province |
ILLINOIS |
| ZIP/Postal code |
60611 |
| Country |
USA |
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| Platform ID |
GPL13776 |
| Series (1) |
| GSE32165 |
Analysis of Caenorhabditis elegans intestinal gene expression and alternative polyadenylation using fluorescence-activated nuclei sorting (FANS) and 3' end deep sequencing (3'end-seq) |
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| Relations |
| SRA |
SRX097314 |
| BioSample |
SAMN00718804 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM797451.bed.gz |
884.2 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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