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Status |
Public on Apr 08, 2009 |
Title |
S2-DRSC-16 |
Sample type |
SRA |
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Source name |
S2-DRSC cell line, biological replicate 2
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Organism |
Drosophila melanogaster |
Characteristics |
cell line: S2-DRSC
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Growth protocol |
Schneider's medium + 10% heat-inactivated FCS
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Extracted molecule |
polyA RNA |
Extraction protocol |
Paired-End RNA-Seq Library Preparation: mRNA Purification from Total RNA Fragmentation of mRNA: Assemble the following reaction: 2 µl 10 x fragmentation buffer (Ambion #AM8740), 11 µl mRNA (100ng/µL), 7 µl Spike-ins. Incubate the tube in a PCR thermocycler at 70°C for 5 minutes, add 1 µL of stop buffer, and put the tubes on ice. Transfer the solution to a 1.5 ml microcentrifuge tube. Add 1 µL of 3M NaOAC, 2 µL of glycogen, and 30 µL of 100% EtOH. Incubate the tube at -80°C for 30 minutes.Spin at 14000rmp for 25 minutes at 4°C in a microcentrifuge. Wash the pellet with 70% EtOH, and air dry the pellet. Resuspend the RNA in 10.5µL of water. First strand cDNA synthesis: Assemble the following reaction: 1.0 µl N6 primer (3µg/µl), 10.5 µl mRNA (100ng/µl). Incubate the tube in a PCR thermocycler at 65°C for 5 minutes, and put the tubes on ice. Mix the following in order, make 10% extra reagent for multiple samples: 4.0 µl 5 1st strand buffer, 2.0 µl 100 mM DTT, 1.0 µl dNTP Mix (10 mM), 0.5 µl (RNaseOUT (40U/µl) Add 7.5µL mixture to the tube, mix well, and heat the sample at 25°C in a thermocycler for 2 min. Add 1 µL SuperscriptII (200U/ µL) to the sample, and incubate the sample in a thermocycler with following program: 10 minutes 25C, 50 minutes 42C, 15 minutes 70C, Hold 4C. Second strand cDNA synthesis: Put the tubes on ice. Add 51µL of H2O to the first strand cDNA synthesis mix. Add the following reagents: 20µL 5 second strand buffer, 3µL dNTP mix (10mM). Mix well, incubate on ice 5 minutes or until well chilled, and add:1 µL RNaseH (2U/µL), 5 µL DNA pol I (10U/µL). Mix well, and incubate at 16°C in a thermocycler for 2.5 hours. Purify the DNA with a Qiaquick PCR spin column, and elute in 30µL of EB solution. Note: The remaining steps use reagents that are part of the Illumina Genomic DNA Sample Prep kit. End Repair: Prepare the following reaction mix: 50ul DNA sample, 25ul water, 10ul T4 DNA ligase buffer with 10mM ATP, 4ul dNTP mix (10 mM), 5ul T4 DNA polymerase, 1ul Klenow DNA polymerase, 5ul T4 PNK (10U/ul). Incubate in thermal cycler 30 min. at 20C. Purify on one QIAquick PCR Purification column (Qiagen part #28104) eluting in 32ul EB. Add 'A' Bases to 3' End of DNA Fragments: Prepare the following reaction mix: 32ul DNA sample, 5ul Klenow buffer, 10ul dATP(1mM), 3ul Klenow 3' to 5' exonuclease. Incubate for 30 min. at 37C. Purify on one Qiaquick MinElute column (Qiagen part #28004), eluting in 19ul EB. Ligate Adapters: Prepare the following reaction mix: 19 ul DNA sample, 25 ul DNA ligase buffer, 1 ul PE Adapter oligo mix, 5 ul DNA ligase (1U/ul). Incubate for 15 min. at room temp. Purify on one Qiaquick PCR purification column, eluting in 10ul EB. Purify Ligation Products: Prepare a 2% agarose gel in a final volumn of 50mL 1x TAE buffer. Load 10µL of the sample into one well, and 1µL 100bp DNA ladder into another. (To prevent cross contamination, leave at least 2 wells between samples). Run gel at 120V for 45~60 minutes. Cut the gel at 300bp (+/- 25bp), and purify the DNA with Qiaquick gel extraction kit (Qiagen part #28604). Elute the DNA into 30µL of EB solution. Enrich Adapter Modified DNA fragments by PCR: Prepare the following reaction mix: 23 ul DNA, 25 ul Phusion DNA polymerase, 1 ul PCR primer 1.1, 1 ul PCR primer 2.1. Amplify using the following protocol= 30 seconds at 98C, 10 seconds at 98C, 30 seconds at 65C, 30 seconds at 72C go to #2 15 more times, 5 minutes at 72C. Hold forever at 4C. Purify with one QIAquick pcr purification column and elute in 30ul EB. Gel purification: Prepare a 2% agarose gel in a final volumn of 50mL 1x TAE buffer. Load the 10µL of sample into one well, and 1µL 100bp DNA ladder into another. (To prevent cross contamination, leave at least 2 wells between samples). Run gel at 120V for 45~60min. Cut the gel at 300 bp (+/- 25bp), and purify the DNA with Qiaquick gel extraction kit. Elute the DNA into 30µL of EB solution. Load onto Solexa flowcell. Dilute sample to 10nM concentration in EB. Load onto flow cell at a 2-4pM concentration.
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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 |
Oligo-dT selected, double stranded cDNA, Paired-end sequencing
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Data processing |
Reads were aligned to the reference genome and a splice junction database to identify uniquely mapping reads and the data converted to GFF files and .bedgraph files which indicate the number of reads that map to each base in the genome.
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Submission date |
Apr 07, 2009 |
Last update date |
May 15, 2019 |
Contact name |
Brenton R Graveley |
E-mail(s) |
graveley@uchc.edu
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Phone |
860-679-2090
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Organization name |
University of Connecticut Health Center
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Department |
Genetics and Genome Sciences
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Street address |
400 Farmington Avenue
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City |
Farmington |
State/province |
CT |
ZIP/Postal code |
06030-3301 |
Country |
USA |
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Platform ID |
GPL9061 |
Series (1) |
GSE15596 |
modENCODE RNA-Seq of Drosophila cell lines Kc167, CME_W1_Cl.8+, S2-DRSC and ML-DmBG3-c2 |
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Relations |
SRA |
SRX003841 |
BioSample |
SAMN02197832 |
Supplementary file |
Size |
Download |
File type/resource |
GSM390064_S2_DRSC_16_spa.bedgraph.gz |
50.8 Mb |
(ftp)(http) |
BEDGRAPH |
GSM390064_S2_DRSC_16_spa.gff.gz |
234.4 Mb |
(ftp)(http) |
GFF |
SRA Run Selector |
Processed data provided as supplementary file |
Raw data are available in SRA |
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