RNA-Seq of WT6 Chlamydomonas reinhardtii R6
Total RNA was extracted from wild type Chlamydomonas grown in the dark for 4 wenks, using the standard TRIzol protocol. For the library preparation, mRNA was processed as previously described, following an adapted version of the single-cell mRNA seq protocol of CEL-Seq (Hashimshony et al., 2012; Simmini et al., 2014). In brief, samples were barcoded with CEL-seq primers during a reverse transcription and pooled after second strand synthesis. The resulting cDNA was amplified by an overnight in vitro transcription reaction. Sequencing libraries were prepared with Illumina Truseq small RNA primers. The DNA library was paired-end sequenced on an Illumina Nextseq 500, high output, with a 1x75 bp Illumina kit (R1: 26 cycles, index read: 6 cycles, R2: 60 cycles). The first read was used to identify the Illumina library index and CEL-Seq sample barcode. The second read was aligned to the C. reinhardtii 281_v5.6 reference transcriptome using BWA MEM (H. Li & Durbin, 2010). Reads that mapped to multiple locations were discarded. Mapping and counting of reads were performed using the MapAndGo script (https://github.com/anna-alemany/transcriptomics/tree/master/mapandgo).