Thermo scientific nanodrop 2000 spectrophotometer

Total RNA was extracted from mouse livers and cultured liver primary and transformed mouse and human cells using miRNeasy kit (Qiagen, USA), and its quantity and quality were assessed using a ThermoScientific 2000 nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). cDNA was prepared using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) and subjected to real-time PCR using gene specific primers. Primer sequences are listed in Table 1. Assays were performed in 96-well PCR plates using All-in-One™ qPCR Mix (Genecopia, USA). The reaction volume of 20 μl contained 10.0 μl SYBR green master mix (2X), 1 μl cDNA, 1 μl of each primer and 7 μl nuclease-free water. The following two-step thermal cycling profile was used (StepOnePlus Real-Time PCR, Life Technologies, Grand Island, NY): Step I (cycling): 95 °C for 5 min, 95 °C for 15 s, 60 °C for 30 s and 72 °C for 15s for 40 cycles. Step II (melting curve): 60 °C for 15 s, 60 °C 1 min and 95 °C for 30 s. The template amplification was confirmed by melting curve analyses. mRNA expression of genes was normalized to 18s expression and fold change in expression was calculated by the 2^–∆∆Ct method.

We used the MoBio Powersoil DNA Isolation Kit (QIAGEN) to extract DNA following the manufacturer's protocol. Sample concentration (ng/µl) and 260/280 ratios were measured using a Thermo Scientific NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). Sequencing libraries of the V4 region (~250 bp) of 16S rRNA were prepared and sequenced by BGI Americas using the llumina MiSeq platform with 250 bp paired‐end reads.

Total RNA was extracted using a MiniBEST Plant RNA Extraction Kit (Takara Biotechnology Inc., Kusatsu, Japan). Approximately 100 mg of fresh mycelia was ground in liquid nitrogen before extraction. The RNA quality and quantity were determined using the Thermo Scientific NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
RNA-seq of the S. bambusicola (GDMCC 60438) mycelia at three different fermentation temperatures was performed by Annoroad Gene Technology Co., Ltd. (Beijing, China), and three replicates for each sample were used for RNA extraction. A cDNA library was constructed for each replicate, and the libraries were sequenced on an MGI T7 platform (BGI Inc., Shenzhen, China) followed by paired-end 150-bp read generation. Then, the low-quality reads were filtered out using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and Skewer (https://sourceforge.net/projects/skewer) with the parameters set to -q 20 -Q 30 -l 50 [27 (link)].

The PCR amplification of the araR gene and its flanking regions for the sequencing experiments and that of the probes for the Southern and Northern blots was performed by using the Phire Hot Start II polymerase (Thermo Scientific, Breda, the Netherlands) according to instructions of the manufacturer using the Prime Thermal Cycler (Techne, (VWR) Amsterdam, the Netherlands) PCR machine. DNA isolation from the fungal mycelia frozen in liquid nitrogen was done according to the procedure described by Arentshorst et al. (2012 (link)) via a phenol-chloroform-isoamyl alcohol extraction. DNA extraction from agarose gels was performed using the GeneJET™ gel extraction kit from Fermentas Life Sciences (Amsterdam, the Netherlands) according to the manufacturer’s instructions. DNA concentrations were measured spectrophotometrically with the Thermo Scientific Nano Drop 2000 Spectrophotometer (Thermo Scientific, Breda, the Netherlands) at a wavelength of 260 nm. RNA extraction and Northern blot analysis were performed as described earlier (Alazi et al. 2016 (link)). Primers for the amplification of the probes are listed in Supplemental Table S1.

Total RNA was extracted from the platelets using a TRIzol reagent (Ambion, United States). The concentration and quality of the total RNA were assessed using Thermo Scientific NanoDrop 2000 Spectrophotometer (Thermo Scientific, United States). Reverse transcription was performed using a PrimeScript RT reagent kit with a gDNA eraser (TaKaRa Bio, Dalian, China) following the manufacturer’s instructions.