Ribo zero plant kit

Total nucleic acid (TNA) extracts were prepared from 22 grapevine accessions as previously described [24 (link)]. TNA was isolated from petioles of selected grapevines. Aliquots of TNA from source samples were subjected to ribosomal RNA (rRNA) depletion and complementary DNA (cDNA) library construction employing a TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina, San Diego, CA, USA). Later, cDNA libraries were sequenced using the Illumina NextSeq 500 platform located at the UC-Davis Genome Center. Sequencing reads were demultiplexed and adapter trimmed via bcl2fastq Conversion Software (Illumina). Trimmed reads were de novo assembled into contigs using SPAdes [25 (link)]. Generated contigs were compared against the complete non-redundant GenBank virus database using BLASTn for nts and BLASTx for aa, providing the annotation used for viral agent identification. Contigs above 500 nts, and with identity over the 80% at the nt or aa level were taken as threshold in order to call the virus species.

Frozen pepper leaf samples were ground using a mortar and pestle in liquid nitrogen. The ground powder (100 mg) was subjected to total RNA extraction using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The quality of the extracted total RNAs was measured using a Technologies 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). The total RNAs with RNA Integrity Number (RIN) values greater than or equal to 7 were used for library preparation. The libraries for RNA sequencing were prepared by depleting ribosomal RNAs from the total RNAs using TruSeq Stranded Total RNA with a Ribo-Zero Plant kit (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions. A total of 15 libraries representing 15 pepper cultivars were prepared and, referred to as P01–P15.

Approximately 150 mg of root tissue was ground in liquid nitrogen, and total RNA was extracted using the Qiagen RNeasy Plant Mini Kit (Valencia, CA, USA). RNA samples were treated with DNase I, evaluated for integrity by agarose gel electrophoresis, and rRNAs were removed by the Ribo-Zero Plant Kit (Illumina, San Diego, CA, USA), and used as templates to construct the library with a ScriptSeq RNA sample preparation kit (Illumina, San Diego, CA, USA). The library was submitted to the W. M. Keck Center, University of Illinois for quality check and cleanup and sequenced on an Illumina HiSeq 4000 for 100 bp paired-end reads.

LncRNAs and mRNAs were isolated from 4-day-old plant seedlings. Ribosomal RNAs (rRNAs) were removed using TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina, 20020610) and the rRNA-free residue was obtained by ethanol precipitation. The first-strand cDNA was synthesized using a random Hexamer-primer and reverse transcriptase (Invitrogen). The second strand cDNA was synthesized using RNase H (Invitrogen) and DNA polymerase I (New England BioLabs). Subsequently, the sequencing libraries were generated using the rRNA-free RNA with a NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, MA, USA), and sequenced by high-throughput sequencing of Illumina Hiseq Xten pair-end 150 (PE150).

Total RNA was extracted from the roots and leaves of CK (abbreviated as CKR and CKL) and Cu-treated samples (abbreviated as CuR and CuL) using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quality and integrity were estimated with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Only high-quality RNA samples (1.8 < OD260/280 < 2.2, OD260/230 ≥ 2.0, RIN ≥ 7.0, 28S/18S ≥ 1.0) were used to construct the sequencing library.
For mRNA, LncRNA, and circRNA sequencing, 5 μg of total RNA was used to prepare ribosomal RNA (rRNA) removed strand-specific library using a TruSeq Stranded Total RNA Library Prep with the Ribo-Zero Plant Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. There were three biological replicates per treatment, and a total of 12 libraries were prepared. For small RNA sequencing, 4 small RNA libraries were constructed with 3 μg of total RNA from CKL, CKR, CuL and CuR samples and the Truseq Small RNA sample prep Kit (Illumina, San Diego, CA, USA). After libraries were quantified by a TBS-380 Fluorometer (Turner Biosystem, Sunnyvale, CA, USA), deep RNA sequencing was performed using an Illumina HiSeq X Ten platform at Shanghai Majorbio Bio-Pharm Biotechnology Co. Ltd. (Shanghai, China).