|
| Status |
Public on Jun 26, 2015 |
| Title |
G. hirsutum 21 day post anthesis cotton fiber biological pool 2 |
| Sample type |
SRA |
| |
|
| Source name |
cotton fiber
|
| Organism |
Gossypium hirsutum |
| Characteristics |
age: 21 days post anthesis
tissue: cotton seed fiber
developmental stage: transition from primary to secondary cell wall deposition
cultivar: DELTAPINE 90
|
| Treatment protocol |
NA
|
| Growth protocol |
Plants were grown in a temperature controlled greenhouse with day/night temperatures of 26/22C and ambient daylength supplemented to 14 hours by a bank of 16 x 500W metal halide lamps. Plants were potted in 10" diameter pots of peat-lite and gravel and were watered with Hoagland's solution once in the morning and once in the afternoon (http://www.ncsu.edu/phytotron/manual.pdf). Plants were rotated weekly to minimize the positional effects within the greenhouse.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Ovules and fibers from first position bolls were removed from cotton bolls, flash frozen in liquid nitrogen and gently ground within 5 minutes of boll dissection. Ovules were removed during the grinding process and the remaining ground fiber was stored at -80C until RNA extraction. Fiber RNA was extracted using the Spectrum Plant Total RNA kit (http://www.sigmaaldrich.com/) and the manufacturer's instructions for protocol A with 750µl of binding buffer and an on-column DNase I treatment using DNASE70 (www.sigmaaldrich.com).
RNA libraries were prepared for sequencing using the Illumina TruSeq RNA sample prep kit v2 (www.illumina.com). Total RNA (1 µg) was purified using poly-T oligo-attached magnetic beads to isolate poly-A containing mRNA. The mRNA was chemically fragmented using divalent cations and fragments were reverse transcribed into first strand cDNA using random primers. Second strand synthesis was carried out using DNA Polymerase I and RNaseH. Ends were repaired on the double-stranded cDNA to convert overhangs into blunt ends, 3’ ends were adenylated, and adapters with indexes for multiplexing were ligated. The library was amplified with a PCR reaction and then small fragments were removed (AmpureXP beads; www.beckmancoulter.com). The final library was quantified on the Agilent Bioanalyzer High Sensitivity DNA Chip; (www.agilent.com) prior to pooling.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Sample name: Gh21p2
Fiber pooled from 3 plants
RNA-Seq of Gossypium hirsutum 21 day post anthesis cotton fiber biological pool 2
|
| Data processing |
Illumina Casava1.8.2 software was used for basecalling and demultiplexing
Raw reads were mapped to the combined cotton A and D EST reference using Burrows Wheeler Aligner version 0.6.2 (http://bio-bwa.sourceforge.net/) allowing maximum 4% mismatch
Mapped reads against individual reference gene were then counted through BEDTOOLS version 2.17.0 (https://code.google.com/p/bedtools/) after file format conversion using SAMTOOLS (http://samtools.sourceforge.net/).
Transcript counts were normalized by RPKM (Reads Per Kilo bases per Million reads) using a PERL script
RPKM values from three technical sequencing replicates per sample were averaged
The samWrapper function in the R DEGseq package (http://bioconductor.org/packages/release/bioc/manuals/DEGseq/) was used to determine differential gene expression
Genome_build: Graimondii2_0 (http://www.ncbi.nlm.nih.gov/assembly/GCA_000327365.1)
Genome_build: Gossypium arboreum transcriptome shotgun assembly - Bioproject PRJNA269608 and TSA accession GBYK00000000
Supplementary_files_format_and_content: Processed data files with read abundances for the inividual samples contain:1) the transcript name (Reference_ID) derived from the Gossypium raimondii genome or a Gossypium arboreum contig assembly (TSA accession GBYK00000000), 2) the mean read count from three technical sequencing replicates normalized as reads per kilobase of transcript per million reads mapped (RPKM), 3)The most homologous gene ID from the Arabidopsis Information Resource (www.arabidopsis.org, Homologous_TAIR_ID), 4) the corresponding Arabidopsis gene name (Arabidopsis_Gene_Name), and 5) the corresponding annotation imported from www.phytozome.net for Gossypium raimondii transcript identifiers or TAIR for Gossypium arboreum contig identifiers (Annotation).
Supplementary_files_format_and_content: Processed data files from differential expression analysis contain: 1) the transcript ID (Reference_ID), 2) fold change ratio for a given comparison derived from the means of three biological replicates (Fold Change), 3) the associated q-value from the pair-wise statistical comparison (q-value), 4) The most homologous gene ID from the Arabidopsis Information Resource (www.arabidopsis.org, Homologous_TAIR_ID), 5) the corresponding Arabidopsis gene name (Arabidopsis_Gene_Name), and 6) the corresponding annotation imported from www.phytozome.net for Gossypium raimondii transcript identifiers or TAIR for Gossypium arboreum contig identifiers (Annotation).
|
| |
|
| Submission date |
Jan 14, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Candace Hope Haigler |
| Organization name |
North Carolina State University
|
| Department |
Crop Science
|
| Lab |
Haigler Lab
|
| Street address |
101 Derieux Place
|
| City |
Raleigh |
| State/province |
NC |
| ZIP/Postal code |
27695 |
| Country |
USA |
| |
|
| Platform ID |
GPL16485 |
| Series (1) |
| GSE64958 |
Transcriptomics and Metabolomics of 10 - 28 day post anthesis cotton fiber from Gossypium hirsutum and Gossypium barbadense |
|
| Relations |
| BioSample |
SAMN03284081 |
| SRA |
SRX745102 |
| SRA |
SRX745103 |
| SRA |
SRX745104 |
