|
| Status |
Public on Jul 25, 2016 |
| Title |
KO_DH |
| Sample type |
SRA |
| |
|
| Source name |
6-day subcultured chloronemata
|
| Organism |
Physcomitrium patens |
| Characteristics |
strain: Gransden
genotype: PpanrKO
|
| Treatment protocol |
For hormone and mannitol treatment, cellophanes were transferred into Petri dishes containing a 3MM paper disc soaked with liquid BCDAT medium containing ABA (10-5M) or mannitol (10% (w/v)) for 1 hour. For dehydration, tissue was transferred from cellophane into empty 9cm Petri dishes and the open dishes were placed in a desiccator over a saturated solution of NaCl, and allowed to slowly dehydrate. Tissue was harvested after 70% of the original fresh weight had been lost.
|
| Growth protocol |
Chloronemal homogenate cultures were grown on BCDAT agar medium overlaid with cellophanes at 25 C, under continuous illumination (50W.m2.cm2) and subcultured every 6 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue was squeezed dry between two sheets of 3MM paper and frozen in liquig N2. Tissue was powdered in liquid N2 and RNA was extracted in 0.1M Tris-Cl, pH9.0-0.5% (w/v) SDS – 2% (w/v) PVP-40 – 5mM beta-mercaptoethanol and emulsified with phenol-chloroform-isoamyl alcohol (25:24:1). Total nucleic acids were recovered by ethanol precipitation and RNA purified by selective precipitation in 2.5M NaCl at 4 C. Residual DNA was removed by digestion with RQ DNase1 at room temperature for 15 minutes, and the RNA recovered by phenol-chloroform extraction and ethanol precipitation.
cDNA libraries were constructed and sequenced by GATC Biotech (Konstanz, Germany). cDNA was synthesised from poly(A)-containing RNA by random primed reverse transcription.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Illumina basecall software
Raw reads were trimmed for low quality bases, adapters as well as first 10-12bp of each read which showed some bias according to fastqc using trimmomatic.
Reads were mapped to the V3.0 Physcomitrella patens genome assembly and to the V3.0 gff3 gene models using tophat2 using the --b2-very-sensitive option.
Mapped reads were sorted and indexed using samtools.
Bedtools was used to extract count data against a gene model gff file of V3.0 gene models for use in Trinity Differential Gene Expression package after TMM normalisation.
Genome_build: Physcomitrella patens V3.0
Supplementary_files_format_and_content: Normalised_abundance.txt data file contains the tab-delimited TMM normalised count values matrix for all libraries with columns in order: WT_C, WT_ABA, WT_DH, WT_man, KO_C, KO_ABA, KO_DH, KO_Man. Pp_genemodels.gff contains a modified version of the full V3.0 gff3 file which contains all gene features. These were stripped out to leave only the gene start and finish coordinates.
|
| |
|
| Submission date |
Sep 01, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrew Cuming |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Leeds
|
| Department |
Centre for Plant Sciences
|
| Street address |
Faculty of Biological Sciences
|
| City |
Leeds |
| ZIP/Postal code |
LS2 9JT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17793 |
| Series (1) |
| GSE72583 |
An ABA response regulator unique to basal land plants required for the acquisition of desiccation tolerance |
|
| Relations |
| BioSample |
SAMN04025437 |
| SRA |
SRX1176831 |
