Truseq rna sample preparation kits v2

Total RNA from the venom gland was extracted using Trizol Reagent (Invitrogen, Waltham, MA, USA), following the manufacturer’s protocol. The RNA concentration and contamination level were measured by UV absorbance using NanoDrop 1000 (Thermo Scientific, Waltham, MA, USA) and RNA integrity was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Next, mRNA was purified from the total RNA using the Dynabeads^® mRNA DIRECT kit (Ambion) and was used to prepare independent cDNA libraries for each venom gland from each snake. The cDNA libraries were prepared following the protocol for TruSeq™ RNA Sample Preparation Kits v2 (Illumina, San Diego, CA, USA) and sequenced using the HiSeq1500 platform (Illumina), generating strand-specific paired-end reads (2 × 150 bp).
We also used the total RNA to sequence miRNAs expressed in the venom gland. The small RNA library was generated using the Illumina Truseq™ Small RNA Preparation kit, following Illumina’s TruSeq™ Small RNA Sample Preparation Guide (Illumina). The purified cDNA library was used for cluster generation on Illumina’s Cluster Station and then sequenced on the HiSeq1500 platform (Illumina).

For the RNA-Seq analysis, the fruit were ground using an homogeniser (Ultra-Turrax T25; Janke and Kunkel IKA-Labortechnik, Staufen, Germany), and the total RNA was extracted from 1 g of the frozen-powder homogenate, according to Landi et al. (2014) (link). The RNA quantity and quality were determined using a Nanodrop 2000 (Thermo Fisher Scientific Inc., Wilmington, DE, USA) and a bioanalyzer (model 2100; Agilent Technologies, Santa Clara, CA, USA). cDNA libraries were prepared from 4 μg total RNA using TruSeq RNA Sample Preparation kits v2 (Illumina, Inc., San Diego, CA, USA), and validated according to the Illumina low-throughput protocol. After normalization, the cDNA libraries were pooled for multiplexing, before loading onto a flow cell (five samples per lane). The hybridization and cluster generation were performed on a cBot System using TruSeq SR Cluster kits v3 (Illumina). The sequencing was performed with an Illumina HiScanSQ platform, using TruSeq SBS kits v3 (Illumina) to obtain single reads 50 nt in length. The indexed raw sequencing reads from each library were de-multiplexed using the CASAVA v1.8 software (Illumina).

A total of 5 × 10⁶ spores/plate of FGSC A1279 and the ΔhosA and ΔhdaA mutant strains were distributed over a CD medium agar plate in duplicate, which was covered with a layer of cellophane and cultivated at 30 °C until the mid-logarithmic phase (48 h). After cultivation, mycelia were harvested and immediately ground to a fine powder in liquid nitrogen and then stored at −80 °C. Total RNA from each sample, with a 28 S:18 S ratio >1.5 and an absorbance OD₂₆₀/OD₂₈₀ ratio between 1.8 and 2.0, was extracted using RNAiso^TM Plus (TaKaRa, Ohtsu, Shiga, Japan) according to the manufacturer’s instructions [14 (link)]. Subsequently, cDNA libraries for each sample were constructed using TruSeq RNA Sample Preparation Kits v2 (Illumina, San Diago, CA, USA) according to the manufacturer’s instructions. After quantification and qualification during the quality control step, the sample library was sequenced on the BGISEQ-500 platform. The raw sequencing data were deposited in the Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/) at the National Center for Biotechnology Information under the accession number PRJNA554537. RNA-Seq data analysis was performed as described in the literature [36 (link)]. The heatmap was plotted using OmicShare Tools, a free online platform for data analysis (http://www.omicshare.com/tools).

RAW264.7, H1819, A7r5, and HEK293 cells were purchased from ATCC^®. A431 cells were a gift of Dr Stanley Cohen (Vanderbilt University, USA). Total RNA from RAW264.7, HEK293, A7r5, H1819, and A413 cells was isolated using TRI REAGENT™ (Sigma-Aldrich) according to the manufacturer’s instructions. RNA quantity was measured with a spectrometer (Nanodrop ND 1000), and RNA quality was analysed on the Agilent 2100 Bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies, USA). We only included RNA samples with an RIN value above 8. Indexed cDNA libraries were generated using TruSeq RNA Sample Preparation kits v2 (Illumina, USA) according to the manufacturer’s protocol. The average library size was 300 bp as determined on the Agilent 2100 Bioanalyzer with DNA 1000 Chips.
The libraries were sequenced on the Illumina HiScanSQ Sequencing System (Interdisciplinary Centre for Clinical Research, Leipzig), generating on average 11.8 ± 1.5 million 101-bp raw paired-end reads per sample on one flow cell lane.