Rna seq libraries

Six Illumina RNA-Seq libraries (one for each organ) were constructed from 500 ng total RNA using the TruSeq Stranded mRNA kit (Illumina, San Diego, CA, United States), which allows for mRNA strand orientation (the orientation of sequences relative to the antisense strand is recorded). Each library was sequenced using 151 bp paired-end read chemistry on a HS4000 Illumina sequencer (Illumina, San Diego, CA, United States).
One Nanopore cDNA library was also prepared from entire female flower RNA. The cDNA library was obtained from 50 ng RNA following the Oxford Nanopore Technologies (Oxford Nanopore Technologies Ltd., Oxford, United Kingdom) protocol “cDNA-PCR Sequencing (SQK-PCS108)” with a 14 cycle PCR (6 min for elongation time). ONT adapters were ligated to 190 ng of resulting cDNA. The Nanopore library was sequenced using a MinION Mk1b and R9.4.1 flowcells.

The quality of RNA from the three biological replicates of each tissue was evaluated by agarose gel electrophoresis, Nanodrop, Qubit, and Agilent 2100. The RNA samples with OD260/280 ≥ 1.8, OD260/230 ≥ 1.8 and 28S/18S ≥ 1.0 were selected for constructing Illumina RNA-seq libraries. The cDNAs of all samples were sequenced using the HiSeq 4000 PE150 platform. The NGSQCToolkit software [30 (link)] was used to eliminate the pair-end reads that contain more than 5% unknown nucleotides or 20% of bases with QS (quality score) < 20. TopHat [31 (link)] was used to align the high-quality reads to the cabbage reference genome. Saturation analysis was performed to reduce the transcription noise. The assembled transcripts generated by Trinity [32 (link)] were mapped to the cabbage reference genome using GMAP [33 (link)].

A total of 111 Illumina RNA-seq libraries of $2\times 75$ nt per read ( $>1800$ million reads) and 5,663,225 GS-FLX RNA-seq single-end reads of Senegalese sole were available from BioProjects PRJNA255461, PRJNA241068 and PRJNA261151^{7 (link)}. These datasets comprised nine experiments that included negative control and drug treatment in triplicate libraries, or even time-course responses. To optimize the number of reads to assemble, only one out of each biological replicate was randomly selected by experiment, and when a time series was available, only the first and the last sampling points were chosen (Supplementary File 1). Libraries containing GS-FLX reads were pre-processed using SeqTrimNext (based on SeqTrim^{67 (link)}), while SeqTrimBB (based on BBmap suite⁶⁸ ) was used for Illumina reads. Default parameters were applied in both cases. Illumina reads shorter than 60 bp were discarded, whereas the threshold for GS-FLX reads was set in 90.
Pre-processed reads (Supplementary File 1) were mapped onto the S. senegalensis genome draft^{33 (link)} using Bowtie2^{69 (link)} with the –no-mixed parameter to discard reads from unpaired alignments. SAMtools^{70 (link)} was used for mapping manipulation and read counts.

In vitro transcription (IVT) RNA was derived from an amplified plasmid library of 1062 human cDNAs (IVT), taken from the Mammalian Gene Collection (Lahens et al., 2014 (link)). Samples were sequenced by two ribosomal depletion protocols polyA selection and Ribo-Zero Gold kit (Epicentre catalog no. RZHM11106). Afterwards the RNA was converted into Illumina RNA-Seq libraries with the TruSeq RNA sample prep kit (Ilumina catalog no. FC-122-1001) and sequenced with an Illumina HiSeq 2000 (paired 100 bp reads). The IVT data have advantages of being a dataset where we know ground truth and it can be sequenced with standard methods, thereby capturing all normal sources of technical error. Importantly, because IVT is efficient, the expression of each base pair is theoretically the same. We used 1062 human full-length cDNAs and performed IVT-Seq. As with simulated data, the full-length transcript forms are known. In this dataset 50 genes had 2 or more splice forms. These ribosomal depletion protocols polyA selection and Ribo-Zero are the two most common protocols, which introduce within-transcript variance (Fig. 3) that cannot easily be simulated.
These data are available at GEO (accessions GSM1219408 for the polyA and GSM1219398, GSM1219399 for Ribo-Zero).