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Protocol publication

[…] RNA was extracted with TRIzol (Life Technologies, Carlsbad, CA) following manufacturer's instructions and treated with DNaseI (Promega, Madison, WI), then cleaned up using Qiagen RNeasy columns (Qiagen, Germantown, MD). cDNA was synthesized using Omniscript (Qiagen, Germantown, MD) starting with 2–3 μg DNA-free RNA per 20 μl reaction. qPCR was performed using a 7300 FAST qPCR machine (Life Technologies, Carlsbad, CA). The primers were designed with a Tm of 60°C using Primer3 or PrimerExpress (Life Technologies, Carlsbad, CA) (). All primer pairs described in this study were confirmed as having 90–105% amplification efficiency and linear amplification within their dynamic range in experimental samples using serial dilutions of cDNA prior to the experiments. Relative transcript levels were calculated by ΔΔCt method () using CβLP as internal reference. [...] RNA was extracted () and the quality was determined using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The triplicate RNA was pooled and 10 μg total RNA was used to prepare RNA-seq library according to the manufacturer's protocol (Illumina, San Diego, CA). The quality of the library was assessed using a 2100 Bioanalyzer before sequencing with Genome Analyzer (Illumina, San Diego, CA). Each sample was run in replicates on two lanes. RNA-Seq data was analyzed as before (). On average, 75% of the sequences could be assigned unambiguously to Augustus v10.2 gene models to generate the matrix of counts per gene. This matrix was used for differential expression analysis using DESeq () using per-condition dispersion estimates and variance stabilization to compute moderate fold changes. Genes were classified as differentially expressed based on a (moderate) twofold regulation and a false discovery rate (FDR) <1%. […]

Pipeline specifications

Software tools Primer3, AUGUSTUS, DESeq
Applications RNA-seq analysis, qPCR
Organisms Chlamydomonas reinhardtii
Chemicals Oxygen