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| Status |
Public on Dec 07, 2012 |
| Title |
Small RNA from fly ovary of w1118: loqsf00791/CyO |
| Sample type |
SRA |
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| Source name |
fly ovary, w1118: loqsf00791/CyO
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| Organism |
Drosophila melanogaster |
| Characteristics |
genotype/variation: w1118: loqsf00791/CyO
gender: female
tissue: ovary
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from 0-10 day female fly heads or 3-5 day ovaries using mirVana (Ambion, Austin, TX, USA). For MEFs, total RNA was isolated using Trizol (Ambion, Austin, TX, USA). The RNA was quantified by absorbance at 260 nm. 18-30 nt RNAs was selected from 100 micrograms total RNA by running a 15% denaturing urea-polyacrylamide gel (National Diagnostics, Atlanta, GA, USA). For fly samples, 2S rRNA was depleted by hybridization using a complementary, immobilized DNA oligonucleotide (5'-biotin-TCA ATG TCG ATA CAA CCC TCA ACC ATA TGT AGT CCA AGC A-3'). Briefly, for each library, 400 µl MyOne Streptavidin C1 Dynabeads (Invitrogen) were washed twice in 0.5× SSC (1× SSC: 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) at 4°C, then re-suspended in 400 µl 0.5× SSC. The beads were then loaded with 200 pmole DNA oligonucleotide at 4°C for 30 min, washed once in 0.5× SSC, re-suspended in 1 ml 0.5× SSC, and then warmed to 65°C for 5 min. Size-selected RNA (50 µg) was incubated at 80°C for 5 min, and then added to the pre-warmed beads and incubated at 50°C for 1 h. The beads were then removed by magnetic capture and the supernatant mixed with 3 volumes of ethanol, 0.3 M (f.c.) sodium acetate, pH 5.2, and 1 µl (20 µg/µl) glycogen (Roche, Indianapolis, IN, USA). 100 pmole of 3' pre-adenylated adapter (5'-rAppTCG TAT GCC GTC TTC TGC TTG T/ddC/-3') was ligated to small RNAs using Rnl2_1-249 (New England Biolabs) at 4 °C for overnight in 20 microliters in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM DTT, 60 mg/ml BSA, and 10% (v/v) DMSO. 3' ligated product was purified from a 15% denaturing urea-polyacrylamide gel (National Diagnostics), and then ligated to 100 pmole of 5' RNA adapter (5'-GUU CAG AGU UCU ACA GUC CGA CGA UC-3' for non-barcoded library and 5'-GUU CAG AGU UCU ACA GUC CGA CGA UCNNNN-3' for barcoded library, NNNN=CGTC, TAGC, ATCC, or GCAC) using T4 RNA ligase (Ambion, Austin, TX, USA) at 4 °C for overnight in 20 microliters in 50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, and 10% DMSO. The ligated product was purified from a 10% denaturing urea-polyacrylamide gel (National Diagnostics). Half the ligated product was used to synthesize cDNA using Superscript III (Invitrogen) and the reverse transcription primer (5'-CAA GCA GAA GAC GGC ATA CGA-3'), and half was used as a minus RT control. The small RNA library was amplified using forward (5'-AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-3') and reverse (5'-CAA GCA GAA GAC GGC ATA CGA-3') primers, and then purified from a NuSieve GTG agarose gel (Lonza, Basel, Switzerland). Purified libraries were sequenced using an Illumina Genome Analyzer II (Illumina, San Diego, CA, USA).
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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 |
OVloqsHypCyOUn
Small RNA
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| Data processing |
The Illumina fastq files generated from the sequencing machine were processed by the following steps:
(1) (Required for libraries with barcodes) Using an in-house C++ script, fastq files were split into 3 or 4 fastq files based on the first 4 nucleotides of each sequence. Sequences whose first 4 nucleotides did not match any barcodes were discarded.
(2) Inserts from each fastq file were retrieved by identifying the first appearance of the first six nucleotides of the 3' adaptor in each sequence. Sequences with 'N' were discarded. Quality strings were ignored.
(3) Same insert sequences were pooled together, and their total number of being sequenced are indicated in the second column of the processed file.
Genome_build: dm3
Supplementary_files_format_and_content: Sequences + number of reads. Barcode and adapter sequences are removed, and the same reads are pooled together.
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| Submission date |
Apr 19, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Ryuya Fukunaga |
| E-mail(s) |
fukunaga@jhmi.edu
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| Phone |
5088561220
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| Organization name |
Umass Medical School
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| Department |
BMP
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| Lab |
Zamore
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| Street address |
364 Plantation st.
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| City |
Worcester |
| State/province |
Ma |
| ZIP/Postal code |
01605 |
| Country |
USA |
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| Platform ID |
GPL9061 |
| Series (1) |
| GSE37443 |
Dicer Partner Proteins Tune the Length of Mature miRNAs in Flies and Mammals |
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| Relations |
| Reanalyzed by |
GSM3277688 |
| SRA |
SRX143457 |
| BioSample |
SAMN00860240 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM919419_OVloqsHypCyOUn.txt.gz |
131.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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