A total of 50 ml of algal culture was sampled for transcriptomic analysis. Total RNA was extracted and sequenced as described by Carrier et al. (2014) (link). A library of RNA-seq was built for each sample. Poly(A) mRNA was isolated from total RNA using oligo (dT) magnetic beads [MicroPoly(A)Purist™ Kit; Ambion]. Two cDNA libraries were built according to the instructions of the manufacturer and sequenced with an Illumina HiSeq 3000 sequencer and with the GenoTool platform. We obtained an average of 2.6.17 and 3,2.17 reads per sample in the CL and DL experiments, respectively.
Raw reads of each sample were filtered using TrimGalore to remove known Illumina adapter sequences. Low-quality reads were excluded using a quality score threshold of 30 and a minimal length of 75 or 150 bases. The quality of reads was assessed using FastQC. Then, sequenced reads for each sample were aligned using Bowtie 2 in the QuasR package in R (Quantify and Annotate Short Reads in R). Each gene within the alignment was counted using the Rsubread package in R with the function feature Counts, based on T. lutea reference genome (Carrier et al., 2014 (link)). Gene counts were obtained for each sample and normalized with DESeq2.
Raw reads of each sample were filtered using TrimGalore to remove known Illumina adapter sequences. Low-quality reads were excluded using a quality score threshold of 30 and a minimal length of 75 or 150 bases. The quality of reads was assessed using FastQC. Then, sequenced reads for each sample were aligned using Bowtie 2 in the QuasR package in R (Quantify and Annotate Short Reads in R). Each gene within the alignment was counted using the Rsubread package in R with the function feature Counts, based on T. lutea reference genome (Carrier et al., 2014 (link)). Gene counts were obtained for each sample and normalized with DESeq2.
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