|
| Status |
Public on Sep 20, 2013 |
| Title |
ssa2D |
| Sample type |
SRA |
| |
|
| Source name |
ssa2D_24C
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: ssa2D
media: YES
stress conditions: unstressed, grown at 24C
|
| Treatment protocol |
The indicated sample was shifted from 24oC to 36oC for 15 minutes
|
| Growth protocol |
Cells were grown at temperatures and in media that are indicated for each sample. For details see Gould KL (2004) Protocols for experimentation with Schizosaccharomyces pombe. Methods 33: 187-188.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Qiagen RNeasy Mini Kit was used to extract total RNA.
1. Poly-A RNA is purified from total RNA (100 ng to 4000 ng) using poly-T oligo-attached magnetic beads. 2. Purified poly-A RNA is eluted from the magnetic beads and is simultaneously fragmented. Cleaved RNA fragments are primed with random hexamers. 3. Randomly primed RNA fragments are used to generate first strand cDNA using reverse transcriptase. 4. The RNA template strand is removed from the first strand cDNA and the second strand cDNA is synthesized. 5. Ampure XP beads are used to purify the ds cDNA from the second strand reaction mix. 6. Overhangs on the double-stranded cDNA are removed using an End Repair mix which results in the generation of blunt ends. A 3' to 5' exonuclease activity in this mix removes the 3' overhangs and a polymerase activity fills in the 5' overhangs. 7. The blunt-ended cDNA fragments are purified using Ampure XP Beads. 8. A single %22A%22 nucleotide is added to the 3' ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. 9. Indexed adapters are ligated to the ends of the ds cDNA. A corresponding %22T%22 residue on the 3' end of the adapter provides a complimentary overhang for ligating the adapter to the %22A%22 overhang on the cDNA fragment. These adapters include all sequences needed for hybridization to a sequencing flowcell . 10. Adapter-ligated cDNA molecules are purified with Ampure XP Beads. 11. PCR is performed to enrich those DNA fragments that have adapter molecules ligated to both ends. The PCR reaction is performed using primers that anneal to the ends of the adapters. A total of 15 cycles of PCR is performed. 12. PCR amplified library molecules are purified using Ampure XP beads. 13. The concentration of the amplified library is measured on a NanoDrop spectrophotometer. 14. An aliquot of the amplified library is run on a DNA1000 bioanalyzer chip to validate the approximate size range of the library. 15. The concentration of cluster forming units in the library is defined by qPCR using the Kapbiosystems KAPA Library Quant Kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Sample 4
|
| Data processing |
Galaxy:FastQC_Remove sequencing artifacts
Galaxy:Tophat Select a reference genome Schizosaccharomyces_pombe_1.1 Is this library mate-paired? single TopHat settings to use full Library Type FR Unstranded Anchor length (at least 3) 8 Maximum number of mismatches that can appear in the anchor region of spliced alignment 0 The minimum intron length 70 The maximum intron length 500000 Allow indel search Yes Max insertion length. 3 Max deletion length. 3 Maximum number of alignments to be allowed 20 Minimum intron length that may be found during split-segment (default) search 50 Maximum intron length that may be found during split-segment (default) search 500000 Number of mismatches allowed in the initial read mapping 2 Number of mismatches allowed in each segment alignment for reads mapped independently 2 Minimum length of read segments 25 Use Own Junctions Yes Use Gene Annotation Model Yes Gene Model Annotations 49: Spombe_chrAll.gtf Use Raw Junctions No Only look for supplied junctions No Use Closure Search No Use Coverage Search Yes Minimum intron length that may be found during coverage search 50 Maximum intron length that may be found during coverage search 20000 Use Microexon Search No
Galaxy:Cuffdiff Transcripts 49: Spombe_chrAll.gtf *from ENSEMBL) Perform replicate analysis No SAM or BAM file of aligned RNA-Seq reads 85: Tophat for Illumina on data 49 and data 31: accepted_hits SAM or BAM file of aligned RNA-Seq reads 77: Tophat for Illumina on data 49 and data 29: accepted_hits Library normalization method geometric Dispersion estimation method pooled False Discovery Rate 0.05 Min Alignment Count 10 Perform quartile normalization No Use multi-read correct Yes Perform Bias Correction Yes Reference sequence data cached Set Additional Parameters? (not recommended) No
Genome_build: Schizosaccharomyces pombe genome build 1.1., for details see Wood V et al. Nature 415(6874):871-880, 2002
Supplementary_files_format_and_content: tabular txt, cuffdiff output, gene expression comparison to control cells
|
| |
|
| Submission date |
Aug 23, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Aleksandar Vjestica |
| E-mail(s) |
aleksandarvjestica@yahoo.com
|
| Organization name |
Temasek Life Sciences Laboratory
|
| Lab |
CDL
|
| Street address |
1 Research Link
|
| City |
Singapore |
| State/province |
Singapore |
| ZIP/Postal code |
117604 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL13988 |
| Series (1) |
| GSE50156 |
Gene expression profiles mas5D, ssa2D, hsf1-overexpressing and heat-stressed wild type Schizosaccharomyces pombe cells |
|
| Relations |
| BioSample |
SAMN02325895 |
| SRA |
SRX338878 |
