Trueseq stranded mrna sample prep kit

In order to extract the total microbial RNA, the modified protocol published previously [19 (link)] was applied. The modifications are described in the Additional file 1. The mRNA library was prepared from samples with the appropriate quality and quantity using the TrueSeq Stranded mRNA Sample Prep kit (Illumina, USA). Sequencing was performed using the Illumina V2 chemistry (2 × 250 bp) and applying the MiSeq paired-end mode.

Synchronized cultures of Sphaeroforma arctica at 12°C were sampled every 6 hr for a total duration of 72 hr. Total RNA was extracted by Trizol and purified using the miRNeasy Mini Kit (QIAGEN) from ~50 mL of culture at each time point. Libraries were prepared using the TruSeq Stranded mRNA Sample Prep kit. Paired-end 50 bp read length sequencing was carried out at the CRG genomics core unit on an Illumina HiSeq v4 sequencer. We obtained between 19 and 32 M reads per sample. Transcripts were quantified using Kallisto software (Bray et al., 2016 (link)) with default parameters. To remove non-expressed genes, we filtered out transcripts that had a mean expression level of <0.5 tpm across all 20 samples. This resulted in a set of 14557 transcripts that were used for clustering.
For S. gastrica and S. tapetis, RNA purification was performed using the RNeasy kit (Qiagen), while for S. nootkatensis, P. gemmata and A. whisleri, RNA was purified using Trizol (Invitrogen, CA, USA). Strand-specific sequencing libraries were prepared using Illumina TrueSeq Stranded mRNA Sample Prep kit and sequenced on an Illumina HiSeq3000 machine (150 bp paired end).

Total RNA was extracted from stem, rhizome, root, or callus tissue using a modified CTAB method [38 (link)]. RNA quality was based on UV absorption ratios, where only samples with ratios above 2.0 (260/280 nm) and 2.2 (260/230 nm) were used. Poly(A) + RNA purification, cDNA library synthesis, emulsion-based PCR (emPCR) and NGS was performed at the McGill University and Génome Québec Innovation Center (Montréal, Québec) as described [53 (link)]. Briefly, RNA quality and quantity was assessed using NanoDrop ND-1000 (Thermo Scientific, Waltham, Massachusetts) and BioAnalyzer 2100 (Agilent Technologies, Santa Clara, California) instruments, and Poly(A) + RNA purification was done using either a Dynabeads mRNA Purification kit (Invitrogen) or TrueSeq Stranded mRNA Sample Prep kit (Illumina, San Diego, California). cDNA synthesis was performed using either a cDNA Rapid Library kit (Roche, Basel, Switzerland) or TruSeq Stranded mRNA Sample Prep kit (Illumina) depending on the downstream NGS method. For Roche-454 GS-FLX Titanium pyrosequencing, data processing was done using GS Run Processor (Roche) to generate Standard Flowgram Format (SFF) files. For Illumina GA and HiSeq sequencing, HCS 1.4 and CASAVA 1.6-1.8 software suites (Illumina) were used to generate raw fastq reads.