Cdna synthesis kit

Total RNA was extracted using the Total RNA Extractor (Trizol) Kit (Sangon Biotech Co., Ltd. Shanghai, China). The quality and quantity of total RNA were analyzed using an UltrasecTM 2100 pro UV/Visible Spectrophotometer (Amersham Biosciences, Uppsala, Sweden) and by agarose gel electrophoresis. The cDNA Synthesis Kit (Illumina Inc., San Diego, CA, USA) was used according to the manufacturer's recommendations to prepare cDNA for library construction and Illumina deep sequencing performed by Sangon Biotech Co., Ltd (Shanghai, China). Alignments were performed using the tool package SOAP2 developed for short oligo nucleotide analysis, allowing up to 2 mismatches with reference sequences. Sequenced reads were aligned to human transcript reference sequences from the ENSEMBL database (Homo_sapiens.GRCh37.55.cdna.all.fa) for expression analysis at gene/transcript levels.

The leaf samples from three independent biological replicates for both CA and control were harvested and frozen in liquid nitrogen. Total RNA of each sample was extracted using the RNAiso kit for polysaccharide-rich plant tissue (Takara, Dalian, China) and purified using DNase1 (TURBO DNase, Ambion, USA) to avoid DNA contamination. RNA Integrity Number (RIN) values (>8.5) were determined using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). The sequencing libraries were constructed using a cDNA Synthesis kit (Illumina Inc., San Diego, CA, USA) following the standard Illumina preparation protocol. Paired-end (2 × 125 bp) sequencing of the cDNA libraries was performed on the Illumina HiSeq 2500 (Illumina Inc., San Diego, CA, USA) by the Biomarker Biotechnology Corporation (Beijing, China). Libraries from each biological replicate yielded more than 6 GB of raw data. The raw reads of transcriptome and their transcript assemblies have been deposited into NCBI Sequence Read Archive (SRA) and Transcriptome Shotgun Assembly Sequence database (Accession: GEZV00000000) under the BioProject number PRJNA 314400.

Five biological repetitions for each genotype at the two growth temperature regimes were used (20 RNAseq libraries). The RNA extractions were performed with 100 mg of powdered tissue using the ConcertTM Kit Plant RNA Reagent (Invitrogen^®) and followed by treatment with the Turbo DNA-free Kit (Ambion^®). RNA integrity and purity were assessed by 1% agarose gel electrophoresis and analyzed on a DeNovix DS-11 spectrophotometer (DeNovix Inc., Wilmington, DE, USA) and a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). All samples presented standard values and RNA integrity number (RIN) higher than 7.0. The TruSeq library preparations were constructed using the cDNA Synthesis kit (Illumina Inc., San Diego, CA, USA). Two lanes of paired-end (2x150 bp) sequencing of the cDNA libraries were performed on the Illumina HiSeq 2000 (Illumina Inc., San Diego, CA, USA). Library preparation and sequencing were performed by AgriLife Genomics and Bioinformatics Services (Texas A&M University, College Station, TX, USA) in April 2015. Sequence cluster identification, quality prefiltering, base calling and uncertainty assessment were done in real-time using Illumina’s HCS 2.2.58 and RTA 1.18.64 software with default parameter settings. All the reactions followed the respective manufacturer’s instructions. Pre-processed libraries are available in SRA under BioProject ID PRJNA609253.

Three biological replicates of P. forbesii flowers were prepared for transcriptome sequencing. Total RNA was extracted from all samples using the RNAqueousTM phenol-free total RNA kit (Ambion, Austin, TX, USA) following the manufacturer’s instructions. The RNA was purified using the RNAClean XP (Beckman Coulter, Inc., Brea, CA, USA) and RNase-Free DNase (QIAGEN, Hilden, Germany) kits. Next, the Nanodrop2000 (Thermo Fisher Scientific, Waltham, MA, USA), Qubit 2.0 (Thermo Fisher Scientific, Waltham, MA, USA), and Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) were used to detect the purity, concentration, and integrity of the RNA samples. Twelve libraries were constructed using the cDNA Synthesis kit following the manufacturer’s protocol (Illumina, San Diego, CA, USA). The sequencing library quality control was performed on the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). The cDNA libraries were pair-end sequenced on Illumina’s HiSeq 2500 platform following the manufacturer’s guidelines.

G. pentaphyllum and G. cardiospermum were collected from two locations of Ankang in Shaanxi province during July 2013 (G. pentaphyllum: 32°25′N, 109°04′E; G. cardiospermum: 32°13′N, 109°01′E), the multiple individual plants mixture including leaves, stems, flowers, shoot tips and developing seeds for each species were frozen immediately in liquid nitrogen, and stored at −70 °C. After mixing an approximately equal weight of mixture for each species, total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer instructions, then poly-A mRNA was isolated from total RNA using poly-T oligo-attached magnetic beads (Illumina Inc., San Diego, CA, USA). The quantity and quality of RNA were assessed by gel electrophoresis and spectrophotometry. Purified RNA was used to construct a directional cDNA library using the cDNA Synthesis Kit (Illumina), and then the cDNA library was sequenced using a HiSeq 2000 (Illumina) to obtain short sequences.

Total RNAs, 10 μg each from germinating conidial spores and mycelia, were used to purify poly (A)+ RNAs using oligo (dT) attached to the magnetic beads (Promega, Madison, WI). RNA samples were reverse transcribed using a cDNA synthesis kit from Illumina (Illumina, Inc. San Diego, CA, USA), and cDNAs of an individual RNA sample were sequenced on Illumina Genome Analyzer II (Illumina, Inc. San Diego, CA, USA) at the DNA Facility, Iowa State University. Over 16 million single reads of 83 bp were generated using Solexa GA pipeline 1.6. No read trimming was performed; only reads with a high percent identity to the reference were selected in read mapping.

Total RNA was extracted from yeast cells using a total RNA Mini Kit (Geneaid) as described [56 (link), 57 (link)]. To remove potential contamination of genomic DNA, total purified RNA was incubated with RQ1 (Promega) DNase at 37°C for 40 min and then inactivated at 65°C for 10 min. Reverse transcription involved a cDNA synthesis kit (Epicentre) in an RNase-free environment. RNA underwent initial incubation at 65°C for 5 min with oligo-dT. Then, the reaction mixture including buffer, dNTPs, DTT, ribonuclease inhibitor, and MMLV reverse transcriptase was incubated at 37°C for 60 min and then inactivated at 85°C for 5 min. cDNA prepared from triplicate biological samples was used for PCR analysis. Real-time quantitative PCR (qPCR) involved use of Stratagene Mx3000p (Agilent Technologies) and Kapa SYBR Fast Universal qPCR Kit (Kapa Biosystems). The primers used in this study are available upon request.