Cfx connect real time qpcr system

Construction of the RNA-seq libraries and sequencing on the Illumina NovaSeq 6000 were performed at the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign. After DNase digestion, purified total RNAs were analyzed on a Fragment Analyzer (Agilent) to evaluate RNA integrity. The total RNAs were converted into individually barcoded polyadenylated mRNA-seq libraries with the Kapa HyperPrep mRNA kit (Roche). Libraries were barcoded with Unique Dual Indexes which have been developed to prevent index switching. The adaptor-ligated double-stranded cDNAs were amplified by PCR for eight cycles with the Kapa HiFi polymerase (Roche). The final libraries were quantitated with Qubit (Thermo Fisher) and the average cDNA fragment sizes were determined on a Fragment Analyzer. The libraries were diluted to 10 nM and further quantitated by qPCR on a CFX Connect Real-Time qPCR system (Bio-Rad) for accurate pooling of barcoded libraries and maximization of number of clusters in the flowcell.

RNA was isolated using a Nucelospin kit (Machery Nagel) according to manufacturer’s instructions. A volume of 1 µg RNA was reverse transcribed using qscript cDNA Synthesis Kit (Quanta Biosciences), and 5 ng cDNA was used as template for amplification. An aliquot of 0.1 µM forward and reverse primers (Supplementary Information) were mixed with PerfeCTa SYBR Green Fastmix (Quanta Biosciences), and SYBR Green signal detected using CFX connect real-time qPCR system (Bio-Rad). Wnt target gene analysis (Figure S4) was carried out using WNT signalling targets (SAB Target List) M96 plates (Bio-Rad) according to manufacturer’s instructions.

DNA was extracted from frozen Liver tissue (20 mg) using DNeasy Blood & Tissue kit (#69504, Qiagen). DNA quality and quantity were estimated using a Nanodrop spectrophotometer (Thermo Scientific). To estimate the amount of mtDNA relative to nDNA, we used two sets of primers encoding a mitochondrial gene (Mt_Nd1 and Mt_Nd6) and a nuclear gene Tubulin (Tuba1) (24 (link)). iTaq universal SYBR® Green supermix (BioRad, USA) and 50 ng of DNA was used and amplification was conducted using CFX Connect Real-time qPCR system (Bio Rad, USA). The relative mtDNA content was calculated using ΔΔCT method (25 (link)). Accuracy of the target amplicon was checked with a melting curve, and each sample was read in triplicate.

Construction of the RNAseq libraries and sequencing on the Illumina NovaSeq 6000 were performed at the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign. After DNase digestion, purified total RNAs were analyzed on a Fragment Analyzer (Agilent) to evaluate RNA integrity. The total RNAs were converted into individually barcoded polyadenylated mRNAseq libraries with the Kapa HyperPrep mRNA kit (Roche). Libraries were barcoded with Unique Dual Indexes (UDI’s) which have been developed to prevent index switching. The adaptor-ligated double-stranded cDNAs were amplified by PCR for 8 cycles with the Kapa HiFi polymerase (Roche). The final libraries were quantitated with Qubit (Thermo Fisher) and the average cDNA fragment sizes were determined on a Fragment Analyzer. The libraries were diluted to 10 nM and further quantitated by qPCR on a CFX Connect Real-Time qPCR system (Bio-Rad) for accurate pooling of barcoded libraries and maximization of number of clusters in the flowcell.

Total RNAs were run on a Fragment Analyzer (Agilent, Santa Clara, CA) to evaluate RNA integrity. The RNAseq libraries were constructed with the TruSeq Stranded mRNA Sample Prep kit (Illumina, San Diego, CA). Briefly, polyadenylated messenger RNAs (mRNAs) were enriched from 500 ng of high-quality deoxyribonucleic acid (DNA)-free total RNA with oligodT beads. The mRNAs were chemically fragmented, annealed with a random hexamer, and converted to double stranded cDNAs, which were subsequently blunt-ended, 3'-end A-tailed, and ligated to indexed adaptors.
Each library was ligated to a uniquely dual indexed adaptor (unique dual indexes) to prevent index switching. The adaptor-ligated double-stranded cDNAs were amplified by polymerase chain reaction (PCR) for eight cycles with the Kapa HiFi polymerase (Roche, CA) to reduce the likeliness of multiple identical reads because of preferential amplification. The final libraries were quantified with Qubit (ThermoFisher), and the average library fragment length was determined on a Fragment Analyzer. The libraries were diluted to 10 nM and further quantitated by qPCR on a CFX Connect Real-Time qPCR system (Biorad, Hercules, CA) for accurate pooling of the barcoded libraries and maximization of number of clusters in the flow cell.

To understand the exercise effect on sepsis, a set of 16 dysregulated genes were selected based on our rat sepsis-CLP model we previously identified with RNA-sequencing (RNA-seq) analysis [25 (link)]. Tissue RNA isolation, cDNA synthesis and real-time RT-PCR (qRT-PCR) analysis were performed as described previously by our lab [25 (link)]. In brief, liver total RNA was extracted using an RNA isolation kit (RNeasy plus mini kit, Qiagen) and the concentrations were estimated with NanoDrop (ThermoFisher Scientific). For cDNA synthesis, two micrograms of RNA was reverse-transcribed using the iScript-Adv cDNA Synthesis Kit (BioRad, USA) and 100 ng of cDNA was used for qPCR analysis in a final volume of 20 μl containing, iTaq universal SYBR^® Green supermix (BioRad, USA) and target specific qPCR gene expression primers (Primer sequence are listed in Additional file 1: Table S1). Amplification was performed using CFX-Connect Real-time qPCR system (BioRad, USA). mRNA expression changes were calculated using the 2 − ΔΔCT method [26 (link)] and expressed as fold-change compared to controls. β-actin was used as a normalization control.