Ribo zero rrna removal kit plant

RNA samples (three biological replicates per tissue), depleted of ribosomal RNA (Ribo‐Zero rRNA Removal Kit Plant, Illumina), were sequenced with Illumina technology (Illumina NextSeq. 500) to obtain an average of 20 million paired‐end reads. We received sequencing raw files containing between 21 million and 60 million paired‐end reads. The raw reads were mapped with the STAR algorithm (Dobin et al., 2013 (link)) against the cassava reference genome (version 6.0). Mapping efficiency was between 67% and 87%. SAM files were converted to BAM files and sorted with the MergeSamFiles tool from the Picard package. Sorted BAM files were used by CuffLinks and CuffDiff algorithms (Trapnell et al., 2012 (link)) to calculate normalized fragments per kilobase per million reads (FPKM) using geometric means of fragment counts across all libraries (Anders & Huber, 2010 ) and the fold changes with corresponding p values. Two different data sets were produced: (i) root‐specific gene analysis; genes not expressed in leaves (FPKM ≤ 3 in all SIL and SOL samples) but in PFR/FR and/or in PSR/SR (FPKM ≥ 5 in at least one PFR/FR or PSR/SR sample) and differentially regulated between PFR/FR and PFR/SR with a foldchange of at least ±2, accepting a p ≤ 0.15. (ii) Gene ontology (GO) analysis; genes differentially regulated between PFR/FR and PSR/SR (p ≤ 0.05) independent of the expression in the leaves.

Total RNA was purified from the 10 potato samples as three biological replicates each that were pooled using the Direct-zol™ kit (Zymo Research, Irvine, CA, USA) with TRIzol reagent based on the manufacturer’s protocol. After purifying total RNA, three quality control (QC) methods were applied: (1) microcapillary electrophoresis with an Implen n50 nanophotometer (Implen, Munich, Germany) for preliminary quantification; (2) agarose gel electrophoresis to assess RNA degradation and potential contamination; (3) Agilent Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA). After the three-step QC, mRNA capturing, cDNA library preparation and sequencing were performed by Novogene (Beijing, China), a genome sequencing company. mRNA from the ten potato samples was enriched using oligo(dT) beads. For long non-coding libraries, rRNA was removed using the Plant Ribo-Zero rRNA removal kit (Illumina, San Diego, CA, USA) using the manufacturer’s protocol, resulting in mRNA (rRNA-depleted RNA-seq).

DNA sequencing was performed using a 800 bp short-insert library on a Illumina HiSeq 2000 platform with a 100 bp paired-end program on half a sequencing lane. For RNA sequencing, total RNA was used to prepare a cDNA library prepared using the Illumina Plant Ribo-Zero rRNA Removal kit and then sequenced on the Illumina HiSeq 2000 platform with a 100 bp paired-end program on half a sequencing lane.