Spin column fungal total rna purification kit

To define the temporal expression patterns of the lincRNAs during wheat-R. cerealis interactions, inoculated leaves were harvested at 2, 4, 6, 8, 10, 12, and 14 dpi. As a control, the hyphae of R. cerealis in vitro (PDA) were collected at the time of inoculation (0 dpi). Specific qRT-PCR primers (see Table S1 in the supplemental material) were designed to distinguish each lincRNA.
A spin column fungal total RNA purification kit (Sangon Biotech, China) was used to extract RNA. qRT-PCR was performed on the CFX Connect real-time system (Bio-Rad, USA) according to a procedure described previously by Yang et al. (58 (link)). The relative expression levels of target lincRNAs in R. cerealis were determined using the 2^−ΔΔCT method (59 (link)); the expression levels of all of these lincRNAs were normalized to the expression level of RcActin. Each sample was analyzed in three biological replicates.

Alternaria sp. MG1 (code: CCTCC M 2011348), a strain previously isolated from the cob of Merlot grape, was used in the study and stored in the China Center for Type Culture Collection (Wuhan, China; Shi et al., 2012 (link)). For preparation of Alternaria sp. MG1 cells, the strain was cultivated in a 250 mL flask containing 100 mL liquid potato-dextrose broth [PDB, 200 g potato (peeled and diced), 20 g glucose, 1000 mL water]. The cultivation was carried out at 28⋅C and 120 rpm in a rotary shaker. After a cultivation time of 21 h, the optimum period previously found to produce abundant resveratrol (Zhang et al., 2013b (link)), the cells were collected by centrifugation at 1136 × g for 10 min at 4⋅C using a refrigerated centrifuge (HC-3018R, Anhui USTC Zonkia Scientific Instruments Co., Ltd., Anhui, China). After washing twice with sterile water, the collected cells were immediately stored in liquid nitrogen until further analysis. Extraction of the total RNA from the cells was performed using a Spin Column Fungal total RNA Purification Kit [Sangon Biotech (Shanghai) Co., Ltd., China]. RNA extraction quality and quantity was analyzed using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Massachusetts, USA) gel electrophoresis, and an Agilent 2100 Bioanalyzer (Agilent Technologies, California, USA).

RNA was extracted from A. brassicae treated with 0, 500 and 1500 μM MT and purified by Spin Column Fungal Total RNA Purification Kit (B518659, Sangon Biotech, Shanghai, China) according to the kit instructions. Steps of mRNA library construction, sequencing, and analyses were performed according to Method S3. Data were mapped to the A. alternate genome: https://ftp.ncbi.nlm.nih.gov/genomes/refseq/fungi/Alternaria_alternata/latest_assemb-ly_versions. The transcriptome raw data (PRJNA 830515) have been deposited in NCBI’s SRA. Gene expression analyses were conducted by qRT-PCR, as described above. Gene information and primer sequences are presented in Table S2.

The UeEgl1 sequence was firstly predicted, by blasting egl1 of U. maydis to U. esculenta whole-genome shotgun sequencing (JTLW00000000.1). Then related primers were designed based on the predicted sequence. All the genomic DNA (gDNA) in this research was extracted by the cetyltrimethylammonium bromide (CTAB) method. Total RNA was extracted and purified using a Spin Column Fungal Total RNA Purification Kit (B518659, Sangon Biotech, China), and reverse-transcribed into complementary DNA (cDNA) with HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R212-01/02, Vazyme, Nanjing). The genomic sequence and cDNA of UeEgl1 were amplified by primers egl1-gF/gR and egl1-cF/cR separately (Table S1). Target sequences were verified by agarose gel electrophoresis and sequencing.

DNA was extracted using CTAB method [35 ]. Total RNA was extracted by Spin Column Fungal Total RNA Purification Kit (B518659, Sangon Biotech, China). cDNA was synthesized by PrimeScript™ II 1st strand cDNA Synthesis Kit (6210A, Takara, Japan). The genomic sequence of UeArginase was identified by PCR-sequencing using the primers Arginase-gF/gR (Additional file 1: Table S1). The open reading frame (ORF) of arginase was amplified by RT-PCR with the primers Arginase-cF/cR (Additional file 1: Table S1). The intron of arginase was verified through comparing the genome sequence and ORF of UeArginase by Clone Manger program. Multiple amino sequence alignment was performed by DNAMAN with clustalW methods. Phylogenetic tree of UeArginase in U. esculenta and related species was constructed with the MEGA 5 programs using the neighbor-joining method.

Tissue samples from wounded, inoculated apples were collected 1 hpi, 3 hpi, and 6 hpi. Samples taken at 0 hpi were used as a control. RNA was extracted strictly according to the operation instructions of Spin Column Fungal Total RNA Purification Kit (Sangon. Co., Shanghai, China). The inoculated apple wound tissues (approximately 2 g) were immediately immersed in liquid nitrogen and ground into a fine powder. Ground tissue samples were placed at −80 °C until further processing. Adsorption columns and collection pipes were used to purify the RNA. The quality of RNA was measured using a One Drop OD-1000+ spectrophotometer (Wuyi Science and Technology Co., Ltd., Nanjing, China). RNA was extracted from the wounds of three independent biological replicates for each time point. Each group was composed of 3 apples.

Total RNA of B. trispora (−) with and without trisporic acids treatment were extracted using a Spin Column Fungal Total RNA Purification Kit (Sangon, Shanghai, China), according to the manufacturer’s procedures. After standard processing procedures, cDNA library were prepared, and the average insert size for the paired-end libraries was 300 bp (± 50 bp). Then, paired-end sequencing was performed on the Illumina HiSeq 4000 platform. Raw RNA-seq datasets of B. trispora are deposited in the sequence read archive (SRA) database (Accession Number PRJNA728833).