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| Status |
Public on Oct 09, 2013 |
| Title |
RNA-seq_spt6I |
| Sample type |
SRA |
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| Source name |
spt6
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: spt6-1; spt6 mutant
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| Treatment protocol |
prototrophic wild-type and spt6-1 strains shifted to 37°C for 2 hours.
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| Growth protocol |
Strains were cultured at 30°C, using Yeast Extract Supplemented (YES) medium
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated from S. pombe cells using a hot phenol method followed by phenol-chloroform extractions, precipitation, and purification using Qiagen RNeasy columns.
10 μg of total RNA were used as starting material for strand-specific library preparation using the Illumina Universal RNA-seq samples prep kit. This kit is an unreleased early version provided by Illumina of the Illumina TruSeq Small RNA Sample Prep Kit. Briefly, poly(A)+ RNA was enriched by two rounds of poly(dT) Sera-Mag magnetic beads purification, then fragmented to an average size of ~200 nucleotides. Fragmented RNA was 3’ de-phosphorylated with Antarctic phosphatase and 5’ phosphorylated with polynucleotide kinase to prepare RNA fragments for subsequent ligation. Illumina RNA adaptors were ligated to the 5’ and 3’ ends using a 3’ RNA ligase and a T4 RNA ligase, respectively. First strand cDNA was produced using a primer specific for the Illumina 3’ adaptor. The library was amplified with 15 cycles of PCR using primers specific for the Illumina adaptors and purified using SPRI-beads (Agencourt, Beckman Coulter). Library size distributions and concentrations were determined on an Agilent Bioanalyzer. RNA-seq libraries were sequenced on an Illumina Genome Analyzer IIx instrument.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
The sequenced reads from each sample were aligned twice using TopHat 77 with default parameters, except setting the intron length range to between 30 and 200 bp, to S. pombe genome assembly ASM294v2, ('-i 30 -I 2000' options). In the first round, no annotation guide was provided.
The splice junctions that were discovered for the four samples were merged with annotated splice junctions (ASM294v2.18) to obtain a complete list of splice candidates. In the second round of alignment, these junctions were provided as a guide to the aligner. ('-j <junction file>, --no-novel-juncs' options).
The reads per kb of exon per million mapped read (RPKM) score was calculated for each transcription unit on the sense and antisense strands; sense scores reflected only exonic reads, whereas the complete transcription unit was used in the antisense direction.
Different samples were normalized relative to each other to account for differences in ratios of antisense and intergenic reads, intron retention rates and t/rRNA filtering efficiency. The RPKM values for each of the samples was scaled by a linear factor, such that the mode of the log fold-change distribution for sense RPKM values between each sample and the average of the four samples fell at 0. The linear factor was between 0.75 and 1.1 for the four samples.
A continuous RNA-seq signal was calculated at single bp resolution for the whole genome as: RNA signal = (number of reads covering the position) / (total number of aligned reads) / (read length in sample) * (normalization factor determined using RPKM values above) * 10^10
Genome_build: ASM294v2
Supplementary_files_format_and_content: wig files show log(RNA-seq coverage signal+1) for either strand at every 10bp
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| Submission date |
Aug 06, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter J Park |
| E-mail(s) |
peter_park@harvard.edu
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| Phone |
617-432-7373
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| Organization name |
Harvard Medical School
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| Department |
Center for Biomedical Informatics
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| Street address |
10 Shattuck St
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL15167 |
| Series (2) |
| GSE49573 |
Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast [RNA-seq] |
| GSE49575 |
Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast |
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| Relations |
| BioSample |
SAMN02303449 |
| SRA |
SRX331955 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1201979_RNA-seq_spt6I_coverage_minus.wig.gz |
1.2 Mb |
(ftp)(http) |
WIG |
| GSM1201979_RNA-seq_spt6I_coverage_plus.wig.gz |
1.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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