Hiseq 2500 v4 system

Total RNA was prepared from 100 mg of funicular tissue using TRIzol Reagent (Sigma–Aldrich, Dorset, UK). Tissue samples were homogenized in 1 mL of TRIzol Reagent and 300 μL chloroform and subsequently precipitated using 500 μL isopropanol (Sigma Chemical, Wicklow, Ireland). RNA samples were stored at −80 °C. Then, 20 μg of total RNA from each sample was treated with RNase-free DNase (QIAGEN, Crawley, West Sussex, UK) to prevent genomic DNA contamination and purified using the RNeasy Mini Kit in accordance with the manufacturer’s instructions (QIAGEN, Crawley, West Sussex, UK). RNA quality and quantity were assessed using automated capillary gel electrophoresis on a Bioanalyzer 2100 with RNA 6000 Nano Labchips, according to the manufacturer’s instructions (Agilent Technologies Ireland, Dublin, Ireland). Then, 5 μg of RNA from each sample was used for library construction using standard protocols. Paired-end libraries were constructed for control funiculi at 28 DAF (CF28) and the funiculi associated with silique walls covered with aluminum foil after 7 days treatment, namely 28 DAF (SF28). The median insert size was 250 bp. Two biological replicate RNA samples from each funiculus were sequenced using the Illumina HiSeq 2500V4 system. The total number of mate-paired reads for each sample ranged from 8,000,000 to 13,000,000. Read lengths of 125 bp were collected.

cDNA was generated with SmartSeq2 protocol (Picelli et al. 2014 (link)) and libraries were prepared with an optimized Nextera protocol (Baym et al. 2015 (link)).
RNA libraries of the 30 samples were pooled and sequencing was performed by the Centre for Genomic Regulation (CRG, Barcelona, Spain). cDNAs were amplified according to the Illumina RNA-Seq protocol and sequenced in three lanes using the Illumina HiSeq 2500 v4 system as paired-end 75-bp reads so that 200-300 million reads per lane (i.e., 20-30 Mio reads/sample) could be achieved.
Quality of the data were checked with FASTQC software 0.11.7 (Andrews 2010 ). Cutadapt 1.18 (Martin 2011 (link)) was used to remove low quality reads (quality-cutoff set to 20) and adapter sequences keeping only paired end-reads having a minimum length of 30 bp. Clean reads were mapped onto the Nile tilapia, Oreochromis niloticus, reference genome (Oreochromis_niloticus.Orenil1.0.92) using Hisat2 2.1.0 (Kim et al. 2015 (link)). Quality control of alignments was ascertained with Qualimap 2.2.1 (Okonechnikov et al. 2016 (link)) and the table of counts was generated with FeatureCounts 1.6.1 (Liao et al. 2014 (link)). The RNAseq produced a total number of clean reads that ranged between 10.06 and 29.2 million reads. About 8.56 to 26.16 million reads were mapped onto the genome.