 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 23, 2013 |
| Title |
0320 CSR-1 IP gfp::cdk-1 |
| Sample type |
SRA |
| |
|
| Source name |
neSi11 gfp::cdk-1; neSi23 flag::csr-1(IP)
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
5' barcode: 5' barcode (D) ATCA
3' barcode: 3' barcode (2) CACTCGGGCACCAAGGA
rna subset: small RNA
|
| Treatment protocol |
Small RNAs of 10-40nt from total RNAs were resolved on a 15% polyacrylamide 7M Urea Gel, along with 2 picomoles of RNA standard (18- and 40-nt) in separate lanes. SYBR gold 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 isopropanol-precipitated with 20mg of glycogen as the carrier. The gel purified RNA was incubated with 0.25 U/µl TAP (Epicentre) and 1U/µl SuperRNaseIN (Ambion) in 10µl reaction containing 1X buffer at 37 oC for 1 hr, followed by phenol-chloroform extraction and isopropanol precipitation. The TAP-treated samples were then incubated with 5µM of 3’-end linker (1, 2, 3 from IDT DNA Technologies), 1 Unit/µl of SuperRNaseIN, 10% DMSO and 1 U/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 5 µM of 5’ adapter oligonucleotide with 4nt barcode at the 5' end, 1 Unit/µl SuperRNaseIN (Ambion) and 2 U/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 HiSeq platform (Illumina, Inc.) and single-end 50 nt reads were obtained.
|
| Growth protocol |
C. elegans worm grown at 20 degree
|
| Extracted molecule |
total RNA |
| Extraction protocol |
small RNA cloning, 5' and 3' ligation dependent
Total small RNA cloning/Small RNA cloning by CSR-1 IP
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
0320 CSR-1 IP gfp::cdk-1
|
| Data processing |
Remove 5' barcode and 3' barcode (or called linker)
Map the reads to WS215 genome and annotation
A custom PERL pipeline is used to summarize reads and generate histogram at single nt level.
Generic genome browse is used to visualize the data
Genome_build: WormBase WS215
Supplementary_files_format_and_content: Tab delimited txt format file containing sequence and read number used in the analysis with the first line as header
|
| |
|
| Submission date |
Aug 04, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Craig Mello |
| Organization name |
University of Massachusetts Medical School
|
| Department |
Program in Molecular Medicine
|
| Lab |
Craig Mello
|
| Street address |
368 Plantatoin Street, Suite AS5-2047
|
| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
| |
|
| Platform ID |
GPL13657 |
| Series (1) |
| GSE49532 |
The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression |
|
| Relations |
| BioSample |
SAMN02302508 |
| SRA |
SRX331486 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1201399_0320csIPcd_processed.txt.gz |
19.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
