|
| Status |
Public on Mar 15, 2012 |
| Title |
Growth-m |
| Sample type |
SRA |
| |
|
| Source name |
Tetrahymena growth cells with medium density, mating type VI
|
| Organism |
Tetrahymena thermophila |
| Characteristics |
mating type: VI
condition: growth
|
| Treatment protocol |
For growth, mating type VI cells at a medium cell density (~200K cells/ml) were collected. For starvation, mating type V or VI cells were starved in 10 mM Tris (pH 7.5) for 3 and 15 hours. For conjugation, mating type V and VI cells that had been starved for 18 hours were resuspended in 10 mM Tris (pH 7.5) at 200K cells/ml and mixed, and samples were collected at 2 and 8 hours after the mixture.
|
| Growth protocol |
Cells were cultured using 1X SPP (1%PP), 30 degrees, 150 rpm shaking.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiashredder (Cat # 79654) and the RNeasy kit (cat. # 74624), from Qiagen. Contains RNA Protect Cell Reagent (Cat. # 76526) and RNeasy Plus Mini Kit (50 samples).
Poly-A mRNA was selected using Dynal magnetic beads (Invitrogen) and fragmented by heating at 94 â. First strand cDNA were synthesized with random hexamer primers and second strand cDNA with DNA polymerase. Double strand cDNA was end-repaired and a single adenosine moiety was added. The Illumina adapters were ligated and gel-electrophoresis was used to select DNA fragments between 200â250 bps in size. Libraries were amplified by PCR with Phusion polymerase. Sequencing libraries were denatured with sodium hydroxide and diluted in hybridization buffer for loading onto a single lane of an Illumina GA flowcell. Cluster formation, primer hybridization and pair-end sequencing were performed using proprietary reagents according to hte manufacturer's recommended protocol (https://icom.illumina.com/).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Data processing |
Sequence image analysis was performed using the Firecrest, Bustard and GERALD programs (Illumina). The low quality bases (Q<5) at the ends of the reads were trimmed. Tophat (Trapnell et al. 2009) were used to map the sequenced reads to the current T. thermophila reference genome (Coyne et al. 2008; October, 2008 release) (ftp://ftp.tigr.org/pub/data/Eukaryotic_Projects/t_thermophila/annotation_dbs/final_release_oct2008/) and find the reads spanning one exon-exon junction. The NUCmer in MUMmer package (Delcher et al. 2002) and a custom script were used to find the reads possibly spanning two or more exon-exon junctions. The mapped reads allowed up to two mismatches and only the canonical GT-AG junction reads were accepted. Transcripts were assembled using the Cufflinks software (Trapnell et al. 2010), and was compared to the predicted gene models using cuffcompare (Trapnell et al. 2010) and custom scripts. Putative ORFs of assembled transcripts were found using GetOrf in EMBOSS package (Rice et al. 2000).
|
| |
|
| Submission date |
Mar 15, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Wei Miao |
| E-mail(s) |
miaowei@ihb.ac.cn
|
| Organization name |
Institute of hydrobiology, CAS
|
| Street address |
donghu south road #7
|
| City |
wuhan |
| State/province |
hubei |
| ZIP/Postal code |
430072 |
| Country |
China |
| |
|
| Platform ID |
GPL9367 |
| Series (1) |
| GSE27971 |
RNA-seq analysis of Tetrahymena thermophila |
|
| Relations |
| SRA |
SRX211444 |
| BioSample |
SAMN01831717 |
