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Sample GSM1955450

Query DataSets for GSM1955450

Status

Public on Nov 30, 2015

Title

tp_stv_h12_r01

Sample type

SRA

Source name

cell culture

Organism

Thalassiosira pseudonana

Characteristics

strain: CCMP 1335
condition: T=12 silicon starvation
replicate: 1

Treatment protocol

750 ml of culture was removed, treated with cycloheximide (final concentration, 20 μg ml-1) and harvested by filtration. Cells were pelleted and stored at -80° C.

Growth protocol

Axenic 8L cultures of Thalassiosira pseudonana (CCMP1335) were grown in artificial seawater medium (NEPC, http://www3.botany.ubc.ca/cccm/NEPCC/esaw.html) at 18C under continuous light (150 μmol/m^2/sec) to a concentration of approximately1x10^6 cells/ml, harvested by centrifugation for 12 min at 3100 x g, and then placed in 8L of silicon free medium in a polycarbonate bottle at a concentration of approximately 1x10^6 cells/ml. Cultures were stirred and bubbled with air under continuous light, and sampled at 0, 4, 8, 12, 18, and 24 hours following inoculation into silicic acid free medium. The -2 hour timepoint was taken two hours prior to silicon starvation.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from cell pellets using TriReagent as described in Hildebrand & Dahlin, 2000.
cDNA libraries were prepared from 4 µg of total RNA for each sample using the Illumina TruSeq Stranded mRNA Sample Prep kit (Illumina, San Diego, CA) according to manufacturer's protocols (Rev. D), which includes a poly-A purification step. The resulting libraries were evaluated for size on an Agilent DNA High Sensitivity chip following the manufacturer's instructions, and quantified by quantitative PCR according to Illumina's protocols. Each library was diluted to 2 nM with 10 mM Tris-Cl, 0.1% Tween 20. Bar-coded libraries were then pooled (10 or 11 per pool), and sequenced on an Illumina HiSeq 2000 with 100 nt paired end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Demultiplexing Custom perl script
Alignment Tophat2 tophat2 --b2-sensitive --solexa1.3-quals --read-realign-edit-dist 0 --no-coverage-search --library-type fr-firststrand --min-intron-length 40 --max-intron-length 2000
Raw counts HTSeq 0.6.1p1 htseq-count --stranded
Normalized counts DESeq2_1.6.3 padj was calculated using the likelihood ratio test with deviance between biological replicates as the reduced model (dds <- DESeq(dds, test = "LRT", reduced = ~replicate))
Genome_build: Thaps3_chromosomes_assembly_chromosomes_repeatmasked.fasta, Thaps3_bd_unmapped_assembly_scaffolds_repeatmasked.fasta (http://genome.jgi.doe.gov/pages/search-for-genes.jsf?organism=Thaps3)
Supplementary_files_format_and_content: csv file containing deseq2 normalized count data for each sample

Submission date

Nov 29, 2015

Last update date

May 17, 2022

Contact name

Raffaela Abbriano

E-mail(s)

raffaela.abbriano@gmail.com

Organization name

University of California San Diego

Department

Scripps Institution of Oceanography