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Sample GSM614253

Query DataSets for GSM614253

Status

Public on Feb 05, 2011

Title

[E-MTAB-5] ILease15_16Spombe_cDNA

Sample type

SRA

Source name

ILease15_16Spombe_cDNA

Organism

Schizosaccharomyces pombe

Characteristics

material type: whole_organism
Sex: mating_type_h_minus
strainorline: JB22 972h-
genotype: wild_type
growthcondition: normal
time: 0h
temperature: 32degree_C
media: YE
culture density: 0.2OD

Extracted molecule

total RNA

Extraction protocol

nucleic_acid_extraction | All cDNA samples used for Illumina sequencing were prepared by first treating ~1 mg of total RNA for 30 min with amplification grade RNase-free DNase (Invitrogen), according to the manufacturers protocols. PolyA enriched RNA was then prepared using an oligo(dT) selection kit (Oligotex Direct mRNA MiniKit, Qiagen). The resulting poly(A)-enriched RNA was then converted to double-stranded cDNA using a cDNA synthesis kit (Superscript choice system for cDNA synthesis, Invitrogen) , primed by an oligo(dT) primer, according to the manufacturers protocols. Random-primed RNA from rapidly proliferating cells was treated with an enzyme that specifically degrades non-capped RNA molecules (mRNA-only, Eukaryotic mRNA isolation kit, Epicentre Biotechnologies) according to the manufacturers protocols. The resulting cDNA was then converted to double-stranded cDNA as above, except that the reaction was primed with random hexamers. RNA samples from the pooled meiotic timepoints were subjected to IVT amplification after undergoing a poly(A) enrichment step as described above. RNA from the oxidative stress condition and prp2 mutant was treated in the same way as the rapidly proliferating samples with oligo(dT)-primed cDNA synthesis after mRNA enrichment.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer

Data processing

Sequence data processing: FASTQ files of sequencing reads were retrieved from the sequencing machine and converted into FASTA files. FASTA sequences were filtered to remove any sequences which were shorter than 15 bp after trimming the sequence from the position of the first N. All remaining FASTA sequences were matched back to the S. pombe genome using BLAT, with tilesize set to 8 and oneoff set to 1, in parallel on the Sanger compute farm. All fasta reads were also matched back to a spliced genome using BLAT (same settings as above), where all known intron sequences were removed. The result files of matches to the spliced and unspliced genome were compiled into a complete and non-redundant set that was used for subsequent analysis.
Sequencing expression scores: Expression scores for every basepair in the genome were assigned based on how many Illumina reads covered each basepair position. The log2 of the score for each nucleotide position was then taken and used for plotting images in R/Bioconductor. The numbers of sequence reads drop towards the 5'-end of long genes. To not bias expression scores against long genes, scores were determined first by taking the sum of the Illumina expression scores for only 300 bp at the 3'-end of each coding region, or the entire length if the coding region was <300 bp, and then dividing by 300 or the length of the coding region if <300 bp.
processed data files:
norm_filtered_wt_current_Set1_solexa_logscore_Rchrm1.txt
norm_filtered_wt_current_Set1_solexa_logscore_Rchrm2.txt
norm_filtered_wt_current_Set1_solexa_logscore_Rchrm3.txt
norm_filtered_wt_current_Set2_solexa_logscore_Rchrm1.txt
norm_filtered_wt_current_Set2_solexa_logscore_Rchrm2.txt
norm_filtered_wt_current_Set2_solexa_logscore_Rchrm3.txt
norm_filtered_wt_current_Set3_solexa_logscore_Rchrm1.txt
norm_filtered_wt_current_Set3_solexa_logscore_Rchrm2.txt
norm_filtered_wt_current_Set3_solexa_logscore_Rchrm3.txt

Submission date

Oct 28, 2010

Last update date

Oct 19, 2011

Organization

European Bioinformatics Institute

E-mail(s)

miamexpress@ebi.ac.uk

Lab

ArrayExpress

Street address

Wellcome Trust Genome Campus