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Sample GSM1419591

Query DataSets for GSM1419591

Status

Public on Nov 01, 2014

Title

SMah37ZM00Ks

Sample type

SRA

Source name

Zn minus

Organism

Chlamydomonas reinhardtii

Characteristics

strain: CC-4532
time point: Zn deficient - t0

Treatment protocol

Zn limitation was induced by inoculating cells from a nutrient-replete culture to a number of 10^5 cells/ml into 100 ml culture medium without supplemented Zn (“first round culture”), and then into medium without supplemented Zn (“second round culture”). ZnCl2 was resupplied at 2.5 µM to these limited cultures where described when they reached a density of 1 x 10^6 cells/ml (“t0h” of zinc resupply).

Growth protocol

Algae were grown from an inoculum of 1 x 10^5 cells/ml in Tris-acetate-phosphate (TAP) medium containing our revised micronutrient composition (Kropat et al., 2011) at 24°C under continuous light (~90 µmol m-2 s-1) with shaking (180 rpm). The desired lighting spectra were achieved by mixing 2 cool white fluorescent bulbs at 4100K for each 1 warm white fluorescent bulb at 3000K.

Extracted molecule

total RNA

Extraction protocol

A volume of culture containing 5 x 10^7 cells was transferred to a 15 ml Falcon tube and centrifuged at 2500 xg for 2 min at room temperature. The supernatant was immediately decanted and the pellets resuspended in 1 ml freshly made lysis buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 20 mM EDTA, 2% sodium dodecyl sulfate). Samples were centrifuged at 600 xg for 3 min at room temperature to pellet starch. The resulting supernatant was snap-frozen in liquid N2 and stored at -80°C for later processing. Upon thawing at room temperature, 10 ml TRIzol (Life Technologies, Carlsbad, California) was added, and the lysates were thoroughly mixed. Samples were incubated for 5 min at room temperature before being transferred to MaXtract HD (QIAgen, Hilden, Germany) tubes. 2 mL of chloroform was added and mixed again. After a 5 min incubation at room temperature, MaXtract HD tubes were centrifuged at 1500 xg for 5 min at room temperature, and the nucleic acid-containing aqueous phase was collected. To extract RNA, samples were processed using the miRNeasy Mini Kit (QIAgen, Hilden, Germany) according to manufacturer’s instructions. To remove contaminating DNA, samples were digested on-column using the RNase-Free DNase Set (QIAgen, Hilden, Germany) according to manufacturer’s instructions. RNA samples were subjected to an ethanol precipitation according to standard laboratory protocol (300 mM sodium acetate and 2.5 volumes of 100 % ethanol) to remove carry-over contaminants from the QIAgen buffers before being resuspended in 50 µL dH2O.
We used the Illumina TruSeq Stranded mRNA Sample Prep LS kit according to the manufacturer's instructions (Protocol Rev. D from SEP-2012).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Basecalls performed using Illumina RTA software.
Basecalls and quality scores were converted to fastq format.
Individual libraries were demultiplexed.
reads filtered for adaptor sequence and low quality calls
alignment to genome by tophat2
expression estimates by cuffdiff
Genome_build: v5.3.1 (Phytozome v9) available at http://genome.jgi.doe.gov/pages/dynamicOrganismDownload.jsf?organism=PhytozomeV9
Supplementary_files_format_and_content: tab delimited file of expression estimate by FPKM for all genes in all samples

Submission date

Jun 24, 2014

Last update date

May 15, 2019

Contact name

Sean D. Gallaher

Organization name

UC Berkeley

Department

California Institute for Quantitative Biosciences

Lab

Sabeeha Merchant