|
| Status |
Public on Sep 25, 2013 |
| Title |
cwf10-ΔNTE _2_2 |
| Sample type |
SRA |
| |
|
| Source name |
S. pombe cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: OHI408
genotype: h- cwf10 2-127-delta ade6-M21X leu1-32 ura4-D18
variation: CWF10.2.127_2
|
| Growth protocol |
RNA was extracted from cwf10+ and cwf10-ΔNTE cells grown at 25°C. Yeast strains were grown in yeast extract (YE) media.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from cells by extraction with hot acidic phenol as described previously (Collart and Oliviero 2001 Curr Protoc Mol Biol).
Replicates were grown together and processed separately for all following steps. The Vanderbilt Technologies for Advanced Genomics Core Facility (VANTAGE, Nashville, TN) used the Illumina TruSeq Stranded mRNA Sample Preparation Kit to convert the mRNA in 100 ng of total RNA into a library of template molecules suitable for subsequent cluster generation and sequencing on the Illumina HiSeq 2500. The input total RNA was quality-checked by running an aliquot on the Agilent Bioanalyzer to confirm integrity. The Qubit RNA fluorometry assay was used to measure concentration. The input to library prep was 50 μl of 100 ng of DNase-treated total RNA (2 ng/μl). The total RNA underwent enrichment of the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the eluted poly(A) RNA was cleaved into small fragments of 120-210 bp using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using SuperScript II reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The cDNA fragments were then put through an end repair process, the addition of a single ‘A’ base, and ligation to the Illumina multiplexing adapters. The products were then purified and enriched with PCR to create the final cDNA sequencing library. The cDNA library underwent quality control by running on the Agilent Bioanalyzer HS DNA assay to confirm the final library size and on the Agilent Mx3005P qPCR machine using the KAPA Illumina library quantification kit to determine concentration. A 2 nM stock was created and samples pooled by molarity for multiplexing. From the pool 12 pM was loaded into each well for the flow cell on the Illumina cBot for cluster generation. The flow cell was then loaded onto the Illumina HiSeq 2500 utilizing v3 chemistry and HTA 1.8. The raw sequencing reads in BCL format were processed through CASAVA-1.8.2 for FASTQ conversion and demultiplexing. The RTA chastity filter was used and only the PF (passfilter) reads were retained for further analysis.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
CWF10.2.127_2 (this part of the paired-end read was not used in this study)
|
| Data processing |
The raw sequencing reads in BCL format were processed through CASAVA-1.8.2 for FASTQ conversion and demultiplexing. The RTA chastity filter was used and only the PF (passfilter) reads were retained for further analysis.
Paired-end reads of 76 base length (each end) originating from each sample were aligned using Bowtie 0.12.7 (Langmead B, et al., 2009 Genome Biol) to the S. pombe genome sequence (Ensembl S. pombe, Build EF1, version 13) (Kersey PJ, et al., 2010 Nucleic Acids Res) as well as to the corresponding exon-exon junctions database (only the first part of the paired-end reads was considered). Up to 3 base-pair mismatches were allowed. Reads that matched multiple loci were removed from further analysis, and the resultant alignment files were processed to generate ‘pile-ups’ against each chromosome. Bowtie commands --sam -p 8 -q -v 3 -m 1
Searches were performed against the genome sequence combined with a dataset of known exon-exon junctions as defined by Ensembl S. pombe release-13. To ensure that a 76 base read mapped to a splice junction, only the last 70 bases of the first exon and the first 70 bases of the second exon were considered (if the exon exceeded length 70). In this way, reads that overlapped a junction by less than 6 nucleotides were excluded. Reads that matched to more than one junction or elsewhere in the genome were also discarded
Genome_build: Ensembl S. pombe, Build EF1, version 58.1a
Supplementary_files_format_and_content: Expression table and RPKM summary for all samples tab-delimited
|
| |
|
| Submission date |
May 31, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Melanie Ohi |
| E-mail(s) |
melanie.ohi@vanderbilt.edu
|
| Organization name |
Vanderbilt University Medical School
|
| Department |
Cell and Developmental Biology
|
| Street address |
465 21st Avenue South
|
| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37232 |
| Country |
USA |
| |
|
| Platform ID |
GPL17225 |
| Series (1) |
| GSE47573 |
Structural and functional characterization of the N-terminus of Schizosaccharomyces pombe Cwf10 |
|
| Relations |
| BioSample |
SAMN02183785 |
| SRA |
SRX288917 |
