Bioanalyzer dna 7500 chip

Sequencing libraries from purified RNAs were prepared using the TruSeq mRNA-Seq kit with associated protocol (Illumina, Inc., San Diego, CA). Briefly, mRNAs were isolated via attachment to oligo(dT) beads, chemically fragmented to a mean size of 150 nt, and reverse transcribed into cDNA via random hexamer priming. Resulting double-stranded cDNA fragments were end-repaired to create blunt-end fragments, 3’ adenine-tailed, ligated with indexed TruSeq adapters (Illumina, Inc.), and PCR-amplified using TruSeq primers (Illumina, Inc.). Purified PCR-amplified libraries were checked for quality and quantity on a Bioanalyzer DNA 7500 chip (Agilent Technologies, Inc.) with 2100 Expert software before normalization and sample pooling. Sample pools of 10 nM were clustered and sequenced on the HiSeq 2000 (WCR) or 2500 (WCR, FAW, SGSB) system with TruSeq Sequencing By Synthesis Rapid v3 (WCR) or v4 (WCR, FAW, SGSB) chemistry (Illumina, Inc.), as per manufacturer’s instructions. Samples were sequenced single-read, fifty cycles per read, to a minimum depth of five million reads per sample and a target depth of ten million reads per sample.

RNA samples were collected from whole bodies of blood fed females at 3 hours and 18 hours post DENV exposure via the RNAeasy Kit (Qiagen) as per manufacturer instructions. A total of 5 samples of 5 pooled females were extracted per each of the 8 treatments. Blood meals were first removed with micro syringe needles with care to leave the midgut intact. RNA was quantified by NanoDrop spectrophotometer (Thermo Fisher Scientific), then verified via Qubit fluorometric quantitation (Thermo Fisher Scientific), and Kapa Library Quantification qPCR assays (Illumina) with RNA integrity assessed via Bioanalyzer DNA 7500 chip (Agilent). The three highest quality samples from each treatment were then selected for Truseq RNA Library Preparation (Illumina) by the Genomics and Bioinformatics Core Facility (http://genomics.nd.edu/genomics-bioinformatics-core-facility) at the University of Notre Dame before transcriptomic analyses on a NextSeq 500 (Illumina).

The three PGPRs, CPCRI-1, CPCRI-2 and CPCRI-3, chosen based on their plant beneficial attributes towards coconut, cocoa and arecanut were grown in Tryptic Soy Broth (TSB) medium at 30°C for 24–48 h. Genomic DNA was extracted using Gen Elute bacterial genomic DNA kit (Sigma, USA) as per the manufacturer's instructions. The extracted DNA was resolved on 0.8% agarose gel to check its integrity. The quality of the genomic DNA samples was assessed using Bioanalyzer DNA 7500 chip (Agilent, CA). The DNA yield was estimated on a TBS-380 Mini-Fluorometer (Turner BioSystems, CA) using PicoGreen dsDNA Quantitation Reagent (Molecular Probes, OR)