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| Status |
Public on Dec 20, 2011 |
| Title |
Asynchronous haploid (second machine run) |
| Sample type |
SRA |
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| Source name |
Schizosaccharomyces pombe cells
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
cell state: Asynchronous; vegetative growth
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| Growth protocol |
Cells were grown in EMM2 media at 25ºC. For pat1.114 induced meioses the appropriate pat1.114 diploid strains were transferred to 32ºC and samples taken before (0) and 3, 5 and 10 hours after the shift.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction: Total RNA was extracted from Schizosaccharomyces pombe cells as described previously (Lyne et al, 2003). Cells were treated with hot acidic phenol-chloroform, followed by phenol-chloroform extraction, RNA precipitation and column purification. RNA quality was determined using an Agilent 2100 Bioanalyser. Ribosomal reduction: Total RNA (9 ug) was "ribosomally reduced" using the Ribominus Eukaryote Kit for RNA Seq (Invitrogen A10837-08) and the Ribominus Concentration Module (Invitrogen K155005). Samples had one or several rounds of ribosomal reduction until they showed a ribosomal reduction of 90% or above on the Agilent Bioanalyser. Libraries were prepared with 500 ng of ribosomally reduced total RNA using the SOLiD Whole Transcriptome Analysis Kit for the first sequencing run; vegetative, IH5974 and IH2912 (before (timepoint 0) and 3, 5, and 10 hours after temperature shift from 25°C to 32°C) (Applied Biosystems 4425680) and the SOLiD Total RNA-Seq Kit for the second sequencing run that included additional 14 samples; vegetative, asynchronous IH5974 (wild type), and a vegetative, asynchronous IH3365 (wild type diploid) alongside temperature shifted: IH2912 (pat1.114 diploid), IH8832 (atf21.delta diploid) and IH8814 (atf31.delta diploid) before, and 3, 5, and 10 hours after a shift from 25ºC to 32ºC to induce meiosis (Applied Biosystems 4445374) with the following changes: RNase III digestion time was increased to 25 minutes, size selection was carried using E-GEL Size Select 2% Agarose Gels (Invitrogen G661002) on the E-GEL iBase power system (Invitrogen G6400UK). Sample barcoding was carried out using the SOLiD Transcriptome Multiplexing Kit (Applied Biosystems 4427046). The libraries were measured on the Qubit Fluorometer (Invitrogen Q3287) using the Quant-IT DSDNA HS Assay Kit (Invitrogen Q32851) and the molarities assessed by a DNA1000 assay (Agilent Technologies) on the Agilent Bioanalyzer (G2938C) before pooling the libraries in equimolar quantities. Sequencing was performed on an AB SOLiD machine (first run 3.0; second run 3.0+).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB SOLiD System 3.0 |
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| Description |
Sequencing was performed using two slides (designated with the suffix S1 and S2, respectievly); SOLiD 3.0+
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| Data processing |
Reads of length 50 bases originating from each sample were aligned using Bowtie (Langmead et al, 2009) to the Schizosaccharomyces pombe genome sequence (Ensembl Schizosaccharomyces pombe, Build EF1, version 58.1a) as well as to its corresponding exon-exon junctions database. Alignments were performed with no mismatches and reads that matched multiple loci were removed from further analysis and the resultant alignment files pre-processed to generate "pile-ups" against each chromosome. Transcription stretches (TBlocks) were detected independently of known annotation using sequence data from samples obtained following the first machine run. Briefly, the algorithm was designed to assemble neighbouring expressed genomic segments into a single transcriptional unit (TBlock) only if those segments were continuously covered by sequence reads across the different samples (with no region larger than 50bp that is not covered).Tblocks were than aligned to the Schizosaccharomyces pombe genome, to define maximum Transcription Start and Termination Sites (TSS and TTS, respectively). The sequencing data was then queried using coordinates from the augmented Schizosaccharomyces pombe annotation including known exons, introns as well as of newly defined untranslated regions (UTRs), anti-sense loci and intergenic TBlocks (UTRs). Using the read counts obtained for a given region, a corresponding expression level was calculated, normalized (by the number of reads in each sample, as well as by the region's length) and log2 transformed. Finally, summarized gene level, antisense, intronic and intergenic TBlocks expression values were reported.
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| Submission date |
Mar 22, 2011 |
| Last update date |
Jun 11, 2013 |
| Contact name |
Danny Asher Bitton |
| E-mail(s) |
d.bitton@ucl.ac.uk
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| Phone |
+44(0)203-108-1604
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| Organization name |
University College London
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| Department |
Department of Genetics, Evolution & Environment
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| Lab |
Genome Regulation/Bähler Lab
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| Street address |
Darwin Building, Gower Street
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| City |
London |
| ZIP/Postal code |
WC1 6BT |
| Country |
United Kingdom |
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| Platform ID |
GPL13315 |
| Series (1) |
| GSE28113 |
Programmed fluctuations in sense/anti-sense transcript ratios drive sexual differentiation in Schizosaccharomyces pombe |
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| Relations |
| BioSample |
SAMN02196860 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM696404_Asy_haploid_SecondRun_S1.bam |
12.2 Mb |
(ftp)(http) |
BAM |
| GSM696404_Asy_haploid_SecondRun_S2.bam |
12.9 Mb |
(ftp)(http) |
BAM |
| Processed data provided as supplementary file |
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