Rnase free dna set

Recombinant DNA techniques were performed according to the general protocols of Sambrook et al. [30 ]. PCR amplifications and hybridisations were performed as previously described [6 (link)]. For measuring expression of the exoA, exoK, exoY2 and exoA genes, quantitative RT-PCR experiments were performed by using primer pairs rt-exoA-F2/R2, rt-exoK-F/R, exoY2rt-F/R, and qnodA-F/R respectively. The S. fredii HH103 16S rRNA was used as an internal control to normalize gene expression (primer pair rt-16S-F2/R2). All primer pairs used are shown in S1 Table. Total RNA was isolated using the High Pure RNA Isolation Kit (Roche) and RNase Free DNA Set (Qiagen) according to the manufacturer’s instructions. This (DNA free) RNA was reverse transcribed to cDNA by using PrimeScript RT reagent Kit with gDNA Eraser (Takara). Quantitative PCR was performed using a LightCycler 480 (Roche, Switzerland) with the following conditions: 95°C, 10 min; 95°C, 30 s; 50°C, 30 s; 72°C, 20 s; forty cycles, followed by the melting curve profile from 60 to 95°C to verify the specificity of the reaction. The fold changes of three biological samples with three technical replicates in each condition were obtained using the ΔΔCt method.

For the two samples, total RNA was extracted by using a PicoPure RNA isolation kit (Acturus) and subjected to DNase treatment (RNase-free DNA set; Qiagen) according to the manufacturer’s protocol. Next, 10 ng RNA was used for cDNA synthesis and amplification by using an Ovation RNA-seq System V2 kit (NuGen Technologies) following the manufacturer’s protocols with minor modifications. The quality and profile of the RNA and amplified cDNA was assessed by using an RNA 6000 Pico Assay Kit with an Agilent 2100 Bioanalyzer, respectively (Agilent Technologies). The cDNA libraries were then further refined by using the Illumina TruSeq RNA Sample Preparation v2 kit (Illumina) with the low-throughput protocol according to the manufacturer’s instructions. Then samples were paired-end sequenced by using an Illumina HiSeq 2500 platform (Illumina Inc.)¹. The average length of the reads was 125 bp.

To help determine how muscle responds to pathogen presence under the five different treatments, we examined the expression of a suite of genes in muscle, CNS and fat body (see Supplementary Materials and Methods). Samples were collected over several weeks, representing different generations of M. sexta. All tissues were handled in accordance with current guidelines for maintaining sample quality (Taylor et al., 2010 (link)). Tissue samples were stored in RNALater at −80°C until extraction.
RNA extraction and real-time quantitative PCR (qPCR) were performed as described previously (McMillan and Adamo, 2020 (link)) and summarized here. RNA extraction was performed using the RNeasy lipid tissue mini kit (Qiagen). All steps adhered to the manufacturer's instructions and included a DNase1 digest (RNase-Free DNaset, Qiagen) step to remove genomic DNA contamination. Concentration and integrity were determined using a Qubit 4.0 fluorometer (Invitrogen, Waltham, MA, USA). Purity was established using an Epoch microplate spectrophotometer (BioTek, Santa Clara, CA, USA). Only samples that adhered to the cutoffs outlined in the MIQE guidelines were used in analysis (Taylor et al., 2010 (link)). For more detailed methodology, please see the Supplementary Materials and Methods.