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| Status |
Public on Jul 17, 2012 |
| Title |
WAGO-9 IP |
| Sample type |
SRA |
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| Source name |
WAGO-9 IP sample
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain background: EG4322
sample type: WAGO-9 IP
age: gravid adults
tissue: adult gonade
5' barcode: (D) ATCA
ip antibody: M2 FLAG monoclonal antibody
ip antibody vendor: Sigma
ip antibody cat. #: F1804
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| Treatment protocol |
normal growth condition
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| Growth protocol |
adult gonade from gravid adults
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| Extracted molecule |
total RNA |
| Extraction protocol |
Small RNAs of 18-40nt were gel purified from a 15% polyacrylamide 7M Urea Gel, along with 20 picomoles of RNA standard (18- and 40-nt) in separate lanes. To make the RNA clonable at the 5' end, either CIP/PNK or TAP treatment is required during cloning, as indicated in the paper. Ethidium Bromide staining was used to visualize the RNA standards. A gel fragment was excised from the sample lanes in the migration range between the two standards. RNA was eluted from the gel fragment in (0.3M NaCl-TE pH7.5) solution overnight and ethanol-precipitated with 20mg of glycogen as the carrier. The gel purified RNA and 1µM of each standard were incubated with 10µM of 3’-end linker, 1 Unit/µl of SuperRNaseIN, 10% DMSO and 3 Units/µl T4 RNA ligase (Takara) in 10µl ligation buffer (50mM Tris-Cl pH7.5, 10mM MgCl2,10mg/mL BSA, 10mM DTT). The 3’ ligated products were gel purified, eluted, precipitated and then incubated with 10µM of 5’ adapter oligonucleotide with 4nt barcode at the 5' end, 1 Unit/µl SuperRNaseIN (Ambion) and 3 Units/µl of T4 RNA ligase in ligation buffer (50mM Tris-HCI pH7.5), 10mM MgCI2, 10mM DTT, 1mM ATP) and 10% DMSO. The ligated products were gel purified and reverse transcribed in a standard 20µl reaction (SuperScript III, Invitrogen). The cDNA was amplified by PCR and purified in a 8% acrylamide gel. PCR products generated for all the samples were sequenced on a Solexa sequencing platform (Illumina, Inc.) and single-end 36 nt reads were obtained.
Synchronous adult flag::wago-9 worms were dounced in a stainless steel homogenizer. FLAG¡ËWAGO-9 was immunoprecipitated from 20 mg of lysate essentially as described (Gu et al., 2009). Small RNAs were extracted from WAGO-9 immune complexes as well as a portion of the input lysate, gel-purified, pre-treated with TAP, cloned and sequenced as described (Gu et al., 2009).
More details in: PMID 19800275
small RNA cloning, 5' and 3' ligation dependent.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
WAGO-9 IP sample
IP(ms13)
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| Data processing |
Samples are prepared using ligation dependent method with 5' linker containing 4nt barcode and 3' linker added to the RNA, and single-end 36nt sequence was obtained using illumina genome analyzer. A custom PERL script is used to de-barcode the sample and remove the 3' linker CTGTAG and beyond. If the read does not contain the complete 3' linker, as represented by CTGTAG, then CTGTA/CTGT/CTG/CT at the very 3' end is removed only once to account for the incomplete 3' linker. RNA at least 17 nt was used in the following analysis.
use bowtie 0.12.7 with parameter v 3 -a --best --strata -m 400 and allow 1 mutation if the RNA size is 19-21, 2 mutations if the size 22-24, 3 mutations if the size is 25 or bigger.
The sample is normalized to 5 million of non-structural reads and the reads of an individual RNA are distributed evenly to all the genomic loci matched
A custom PERL pipeline is used to perform the post-bowtie analysis, including summarizing, making gff, drawing scatter plot, grouping reads, etc
Genome browse 1.70 plus custom perl script are used to visualize the RNA matched
Genome_build: Wormbase WS215
Supplementary_files_format_and_content: Fasta format sequence with id containing the RNA number matched to the genome and transgene (The 'x' and the number following are the RNA counts and the number before the x is the id)
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| Submission date |
Jun 14, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Craig Mello |
| Organization name |
University of Massachusetts Medical School
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| Department |
Program in Molecular Medicine
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| Lab |
Craig Mello
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| Street address |
368 Plantatoin Street, Suite AS5-2047
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| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
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| Platform ID |
GPL9269 |
| Series (1) |
| GSE38724 |
piRNAs initiate an epigenetic memory of non-self RNA in the C. elegans germline. |
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| Relations |
| SRA |
SRX154631 |
| BioSample |
SAMN01048331 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM948685_wago9IP_processed.fa.gz |
7.9 Mb |
(ftp)(http) |
FA |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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