Nebnext ultra directional rna library preparation kit

Total RNA samples were processed twice with MICROBEnrich and MICROBExpress bacterial mRNA enrichment kits (Ambion/Life Technologies, Grand Island, NY, USA) to remove eukaryotic mRNA and 16S/23S rRNAs, respectively. The purified mRNA was resuspended in 25 µL of nuclease-free water. The quality of enriched mRNA was analyzed using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Next, we generated cDNA libraries from 100 ng of enriched mRNA using the NEBNext Ultra directional RNA library preparation kit and NEBNext dual index oligonucleotides for Illumina (New England BioLabs, Ipswich, MA, USA). To ensure proper enrichment of bacterial transcripts, we first sequenced the pooled library using a MiSeq Reagent Nano Kit v2 (300 cycles) at Clemson University. Deep sequencing of the library was next performed by the NextGen DNA sequencing core laboratory of the Interdisciplinary Center for Biotechnology Research at the University of Florida (Gainesville, FL, USA). Samples were sequenced on the NovaSeq 6000 platform on a single S4 flow cell using a paired-end 2 × 150 bp chemistry.

B. saltans cells were treated with gentamicin (50 μg/ml) before RNA extraction to get rid of extracellular bacteria. Total RNA was extracted from cells and rRNA depleted using RiboZero rRNA depletion kit (Epidemiology). A strand-specific library was constructed with the NEBNext Ultra Directional RNA library preparation kit (NEB, UK). Paired end 2 × 150 bp sequencing was carried out on the HiSeq 4000 platform (Illumina, USA). Raw reads were mapped on to the endosymbiont genome sequence using bwa v0.7.12 and low quality read alignments were removed using samclip (https://github.com/tseemann/samclip) and default parameters. Sort read alignments were visualised with the IGV 2.8.6 tool, while strand-specific read quantitation was performed with the SeqMonk program (https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/). To account for the presence of low DNA contamination in our library, a Difference Quantitation correction was applied. Read counts for each gene were calculated as the read counts originated from the opposite strand (template strand) minus the read counts from the same strand (coding strand) and normalized to the length of each gene (RPK).

The total RNA of tissues was extracted using the Trizol (Thermo Scientific, Wilmington, United States) kit. Electrophoresis using agarose gels at a concentration of 1% to detect RNA degradation and contamination. RNA purity testing was performed using the Kaio K550 (CAIO, Beijing) spectrophotometer. The Qubit^® RNA Detection Kit in the Qubit^® 2.0 Fluorometer (Life Technologies, California, United States) is used to determine RNA concentration. RNA nano 2100 assay kit from the Agilent Bioanalyzer 6000 System (Agilent Technologies California, United States) for RNA integrity testing.
Take 3 μg of total RNA to construct the library. First, Ribo-ZeroTM GoldKits (Epicentre, United States) was used to remove ribosomal RNA (rRNA). In addition, RNA sequencing libraries were generated according to the manufacturer’s instructions for the Illumina NEB Next Ultra Directional RNA Library Preparation Kit (NEB, Ipswich, United States). Sequencing was then performed on the Hiseq X (Illumina, San Diego, CA, United States) platform.

Total RNA from S2 cells was extracted using TRIzol reagent (Thermo Fisher Scientific) and then isolated from the aqueous phase using the EZNA total RNA kit I (Omega Bio-tek). The following steps were performed by the Cornell RNA Sequencing Core (Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University). Poly(A)⁺ RNA was isolated with the NEBNext Poly(A) mRNA magnetic isolation module (New England Biolabs). TruSeq-barcoded RNA-seq libraries were generated with the NEBNext Ultra Directional RNA library preparation kit (New England Biolabs). Each library was quantified with a Qubit 2.0 (dsDNA HS kit, Thermo Fisher Scientific), and the size distribution was determined with a fragment analyzer (Advanced Analytical) prior to pooling.

Genomic DNA was isolated from roots and leaves of the cultivar Charentais (Cucumis melo L. subsp. melo var cantalupensis) using E.Z.N.A Plant DNA Kit (Omega). Fragmentation was performed using Diagenode Bioruptor 200 UCD-300 (30 s then off 90 s for 25 cycles, low power position). The following steps were performed using Diagenode Auto hMeDIP Kit in the SX-8G IP-Star® Compact System. Anti-5-methylcytosine antibody (NA8133D3, Merck Millipore, Diagenode) was used for precipitation. DNA was then purified using Auto Ipure kit v2 (Diagenode). Libraries were synthetized using NebNext Ultra DNA Library Preparation Kit (NEB) according to the manufacturer’s instructions and sequenced by Illumina technology. The same melon accession was used to collect the the total RNA from roots of 10 day old (cultivated in vitro) and from leaves of 3 weeks old, grown in a growth chamber (long day conditions, temperature: 27 °C (day) and 21 °C (night), relative humidity: 60%). The Nucleospin RNA kit (Macherey-Nagel) was used for the extraction according to the manufacturer’s instructions. Libraries were synthetized from 2 µg of total RNA using NEBNext Ultra Directional RNA library Preparation Kit (NEB) according to the manufacturer’s instructions. Two biological replicates were analysed for each tissue. Libraries were sequenced by Illumina technology.