|
| Status |
Public on Sep 12, 2009 |
| Title |
Pupae_combined_RNAseq_1,2 |
| Sample type |
SRA |
| |
|
| Source name |
Pupae
|
| Organism |
Drosophila melanogaster |
| Characteristics |
development stage: Pupae
|
| Treatment protocol |
No Treatment
|
| Growth protocol |
y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder. Embryos and larvae are collected using a filter mesh and a brush. They are rinsed with EWB and crushed in Trizol reagent. Pupae are collected on the side of a vial with a brush. Adult animals are sorted by sex on a CO2 pad and, rinsed before crushed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Matching total RNA was collected from each time-point of this study in TRIzol reagent and isolated according to the manufacturer instructions. DNAse I treated RNA is then purified and concentrated using the RNeasy MinElute Cleanup Kit. PolyA RNAs are then purified using the micropolyA purist Kit from Ambion and converted into single stranded DNA after Reverse Transcription using random hexamers. The second strand synthesis is then carried out by adding to the reaction the RNAseH (Invitrogen #18021014) and DNA Polymerase II (NEB #M0209S) enzymes. At this stage, the double stranded DNA is then cleaned up on Qiagen Quiaquick columns and the ends are repaired by using the T4DNA Polymerase, Klenow Fragment and T4 PNK enzymes.
After another round of purififcation, an A residue is added by the use of the Klenow [3'>5' exo-] enzyme and again purified on Quiaquick columns. Adapters from Illumina for LM-PCR are then ligated to the end of the DNA molecules. The product of the reaction is then run on an Agarose gel (2% NuSieve) and a band corresponding to 100 bp is then extracted and purified. 20 cycles of PCR reaction using the phusion polymerase (Finnzyme F-530S) enzyme and the Illumina oligos are then performed and the product is again purified on gel. Solexa sequencing is then performed on Genome Analyzer with standard Illumina 36 cycles reaction kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
combined_RNAseq_1 and combined_RNAseq_2
|
| Data processing |
Reads were aligned to the flybase BDGPv5 reference genome using TopHat (http://tophat.cbcb.umd.edu/). The bed file was generated by thresholding the bedgraph file (tag count>=1; minimum run length=50; maximum gap=50).
|
| |
|
| Submission date |
Sep 11, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Kevin P. White |
| E-mail(s) |
kpwhite@uchicago.edu
|
| Organization name |
University of Chicago
|
| Department |
Institute for Genomics and Systems Biology
|
| Street address |
900 E. 57th STR. 10th FL.
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60615 |
| Country |
USA |
| |
|
| Platform ID |
GPL9058 |
| Series (3) |
| GSE15292 |
Drosophila at different time points of development: ChIP-chip, ChIP-seq, RNA-seq |
| GSE18068 |
Genome-wide transcriptome sequencing at different stages of Drosophila development, RNA-seq |
| GSE18572 |
Discrete genomic domains exhibit co-occurrence of H3K9me3 and H3K27me3 histone modifications throughout Drosophila |
|
| Relations |
| Reanalyzed by |
GSM3274540 |
| SRA |
SRX012984 |
| BioSample |
SAMN00005133 |
| Named Annotation |
GSM451813_Pupae_combined_coverage.bed.gz |
