Truseq sample prep kit

Root zones pooled from 30 plants (an average) were collected separately into plastic tubes with liquid nitrogen and stored at −80 °C. Total RNA was isolated from plant samples using the Trizol extraction method combined with RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. Residual DNA was eliminated by treating the samples with DNAse I using the DNA-free kit (Ambion). The RNA quantity and quality were confirmed spectrophotometrically with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and using 1% agarose gel electrophoresis. For whole-transcriptome sequencing, cDNA libraries from the total RNA of root segments were prepared with a TruSeq Sample Prep Kit (Illumina) according to the manufacturer’s instruction. Sequencing was performed using an Illumina HiSeq 2500 instrument (Illumina) with single-end 60 bp reads. Data in the form of raw reads and sample preparation descriptions were deposited at NCBI Sequence Read Archive (SRA) and are available at BioProject ID PRJNA639682. All samples were analyzed using RNA-Seq analysis in two independent biological replicates.

The electric lobe from a female marine ray Tetronarce californica(Aquatic Research Consultants; San Pedro, CA) was dissected from the central nervous system, and total RNA was isolated from the frozen tissue as described [45 (link)]. RNA concentration was determined using a Nanodrop spectrophotometer and quality assessed by denaturing gel electrophoresis in formaldehyde gels and Northern analyses [45 (link)]. Poly(A)⁺RNA was purified using Poly(A)Purist MAG Kit (Ambion) and further cleaned up with Turbo DNase (Ambion) and Terminator-5’-phosphate-dependent exonuclease (Epicentre) to remove any trace amount of DNA and rRNA. cDNA was synthesized from purified poly(A)⁺mRNA using random hexamer oligonucleotide or oligo dT as primer. Synthesized cDNA was then sheared into small pieces of 100 to 800 bp in length using a Biorupter. The fragmented cDNA was prepared for Illumina sequencing using TruSeq Sample Prep Kit (Illumina). Paired-end sequencing (100 bp) of the cDNA libraries was performed on a HiSeq 2500 instrument (Illumina).

Formaldehyde-crosslinking of cells, sonication, DNA extraction of FAIRE-enriched fractions and Illumina library preparation was performed as previously described [19 (link)]. Libraries were prepared in triplicate from infected and mock-infected samples at 1, 12, 24 and 48 h, using the Illumina TruSeq Sample Prep kit, and were sequenced on the Illumina 2500 platform (101 bp paired-end read protocol) at the Genome Resource Centre, Institute for Genome Sciences, University of Maryland School of Medicine. Sequence data are available from the NCBI GEO archive GSE132448.

Genomic DNA from the cells was extracted using YeaStar DNA kits (Zymo Research, Irvine, CA, USA) and purified using Genomic DNA purification kits (Promega, Madison, WI, USA) for genome sequencing. DNA quantity and quality were confirmed using the Quant-It PicoGreen dsDNA assay kit (Invitrogen, Waltham, MA, USA) and agarose gel electrophoresis, respectively. Barcoded library construction and genome sequencing using Illumina Miseq instrumentation (Illumina, San Diego, CA, USA) were performed at CHUNLAB (CHUNLAB, Seoul, Korea). Barcoded shotgun genomic DNA libraries were constructed using the TruSeq Sample Prep Kit, following the manufacturer’s instructions (Illumina), and were subjected to 300 base pair (bp) paired-end sequencing by Illumina Miseq. Sequencing raw data were deposited in the Sequence Read Archive and are available at the NCBI BioProject PRJNA690663. SNP analyses were performed using CLC Genomics Workbench version 5.1 (QIAGEN, Hilden, Germany). Reads were trimmed based on quality scores from default program settings. The reads were mapped to an S288C yeast reference sequence (obtained from Genbank) to identify SNPs in SR8LDH and BK01. Next, unique, non-synonymous SNPs in BK01 relative to SR8LDH were identified (Table S1).