Rnase free dnase set

The RNA of PK-15 cells were lysed with Trizol^® Reagent (Invitrogen, United States) according to the instructions. The total RNA contaminating DNA was degraded by treating each sample with RNase-Free DNase Set (Sangon Biotech, China). After purification, total RNA was quantified by optical density (Q3000, Quawell, United States) and the quality was evaluated by gel electrophoresis. The first cDNA was synthesized by HiScript II 1st strand cDNA synthesis kit (Vazyme, China). All primers of target genes were commercially synthesized by Tianyi huiyuan biotechnology company, China (Table 1). The conditions of RT-qPCR were conducted by ChamQ SYBR Color qPCR Master Mix (Vazyme, China) according to the manufacturer’s instructions. The mRNA and miRNA levels were normalized against the amount of the housekeeping gene transcript β-actin and U6, respectively. And the relative expression was calculated by the 2^-ΔΔCt method (Wang X. et al., 2012 (link)).

Total RNA was isolated from R. cellulolyticum cultures on cellobiose in mid-log-phase, and genomic DNA was removed using RNase-Free DNase Set (Sangon, China). The RNA quality was determined using a NanoVue Plus spectrophotometer (Biochrom, UK), and electrophoresis on 1% agarose-formaldehyde gels was performed.
Two micrograms of total RNA were electrophoresed on 1% agarose-formaldehyde gels and blotted onto a positively charged nylon membrane (GE HealthCare, USA) using the NorthernMax kit (Life Technologies, USA). DIG-labeled DNA probes for the detection of specific transcripts were generated with a DIG labeling and detection kit (Roche, Switzerland), as per the manufacturer’s instructions, using the oligonucleotides listed in Table S5.

Total RNA was isolated using a Fungal RNA Kit (Omega) and purified with RNase‐free DNase Set (Sangon) following the manufacturer's instructions. RNA was quantified using Eppendorf Bio Photometer Plus. Total RNA was reverse transcribed to complementary DNA (cDNA) using M‐MLV reverse transcriptase (Promega) according to the manufacturer's instructions. Gene expression was measured by real‐time qPCR with SYBR Green I (Takara) using the IQ5 real‐time system (BioRad) following the protocol described previously (Gu et al., 2014). Data were analysed using the 2^−ΔΔCt method with β‐tubulin as an internal control (Bustin et al., 2009). All assays were performed in three independent replicates and expressed as the mean ± SEM. Data were analysed using GraphPad Prism v. 5.0 software. Primers for each gene used in real‐time qPCR are listed in Table S1.
Genetic information for each gene was obtained by BLAST searching in the S. turcica database (http://genome.jgi.doe.gov/Settu1/Settu1.home.html) using the query homolog gene in S. cerevisiae from the Saccharomyces Genome Database (https://www.yeastgenome.org/). Information for the genes examined in this study is listed in Table S2.