|
| Status |
Public on Jan 10, 2013 |
| Title |
3tfill_ypgal_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
mid-log phase cells, OD 1
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: SLS045 (diploid, S288c background)
media: YPGal media
forward side barcode: None
reverse side barcode: GCCTAA(T)
protocol: 3tfill
|
| Growth protocol |
Yeast cells were grown to mid-log phase (OD1) using either YPAD (1% yeast extract, 2% peptone, 1% glucose) or YPGal (1% yeast extract, 2% peptone, 1% galactose).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by a standard hot phenol method and contaminant DNA was removed by DNase I treatment. The sample was subject to different alternative transcript end or poly(A) site mapping protocol in order to map the poly(A) sites of each transcript. 3T-fill: Starting from total RNA, poly(A) RNA is reverse transcribed using a biotinylated adapter containing an anchored oligo-dT primer. After second strand synthesis, fragments are captured by streptavidin beads, end-repaired, adenylated, and ligated to a second adapter The final library is then loaded into an Illumina cluster station where the poly(A) stretch is filled in with dTTPs before the sequencing reaction starts. This enables the sequencing to start immediately after the poly(A) tail. Internal: The library was prepared as the 3T-fill but using different oligos that allows the sequencing into the poly(A) tail (from the body of the cDNA) and omitting the T-fill step on the illumina clustering station. In this case a stringent library size selection (200bp was performed). BpmI: The library was prepared as the previous one but, introducing a restriction site on the priming oligo that allows the cleavage off the poly(A) tail. RNASeq: strand specific RNASeq protocol usong dUTP digestion as previously described (PMID:21943893). Yoon: RNA-Seq protocol enriching for poly(A) containing fragments after RNA fragmentation, as previously descrived (PMID:20421314).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
B.subtillis capped IVT spike-ins added
|
| Data processing |
Base-calling was performed using Illumina CASAVA pipeline and standard parameters.
3' sequences were extracted from demultiplexed sequencing reads for poly(A) site mapping protocols.
Reads were aligned to the S288c reference R63 genome assembly using the GSNAP aligner.
Transcript poly(A) sites were identifed from data as the base before the start of the poly(A) tail.
Reads and poly(A) sites were mapped to the annotated S.cerevisae transcriptome from Xu et al. 2009 lifted-over to match with genome version (very minor changes)
Genome_build: Saccharomyces cerevisiae S288C reference genome R63 downloaded from SGD(http://www.yeastgenome.org/)
Supplementary_files_format_and_content: Bedgraph and gff files contain poly(A) site calls from the 3'T-fill protocol.
|
| |
|
| Submission date |
Aug 14, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Aino Inkeri Järvelin |
| Organization name |
University of Oxford
|
| Department |
Department of Biochemistry
|
| Street address |
South Parks Road
|
| City |
Oxford |
| ZIP/Postal code |
OX1 3QU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL13821 |
| Series (1) |
| GSE40110 |
Saccharomyces cerevisiae 3' poly(A) site mapping |
|
| Relations |
| SRA |
SRX176616 |
| BioSample |
SAMN01113248 |
