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[…] . A total of 30 μg mixed RNA from three biological replicates detected by 2100 Bioanalyzer (Agilent, USA) was digested with DNase I (TAKARA), and then purified by Dynabeads® Oligo (dT)25 (Life, USA). 100 ng derived mRNAs were fragmented and reverse transcribed into first-strand cDNAs with random hexamer and then the second-strand cDNAs were synthesized by using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina (NEB). The double-stranded cDNAs were purified and ligated to adaptors for Illumina paired-end sequencing. The cDNA library was sequenced using the Illumina HiSeq2500 system by Shanghai Hanyu Biotech lab (Shanghai, China)., For the assembly library, raw reads were filtered using the FASTX-Toolkit ( to remove adapters and low-quality reads (base quality < 20, read length < 40 bp). The obtained quality-filtered reads were de novo assembled into contigs by the Trinity Program []. Unigenes were defined after removing redundancy and short contigs from the assembly. The unigenes were predicted by “GetORF” in the EMBOSS package [] and aligned to the protein sequence database NCBI NR (non-redundant protein database), Swiss-Prot (Annotated protein sequence database), KEGG (Kyoto encyclopedia of genes and genomes) and COG (Clusters of orthologous groups of protein) by Blastp with an E-value threshold of 1 × 10 −5., The number of unique-match reads was calculated and normalized to RPKM (reads per kb per million reads) for gene expression analysis. Comparison of unigene expression between treatments was according to DESeq as described by Abders and Huber []. The differentially expressed genes (DEGs) between NC and A84, or between NC and A840, or between A84 and A840 were restricted with FDR (false discovery rate) ≤ 0.001 and the absolute value of log2 Ratio ≥1., To examine the expression profile of DEGs, the expression data υ (from NC, A84 and A840 treatment) were normalized to 0, log2 (υA84/υNC), log2 (υA840/υNC), and then clustered by Short Time-series Expression Miner software (STEM) []. The clustered profiles with p-value ≤ 0.05 were considered as significantly expressed. Then the DEGs in all or in each profile were subjected to gene ontology (GO) classifications using WEGO [], and KEGG pathway enrichment analysis., The cDNA was generated from 1 μg total RNA isolated from the fronds using a Prime-Script™ 1st Strand cDNA Synthesis Kit (TAKARA, Japan). Primers for quantitative real time PCR (qRT-PCR) were designed using Primer Premier 5.0 software (Premier, Canada) and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The 18S (GenBank accession number: KJ400889) was selected as reference. All the primers are shown in Additional file : Table S1. qRT-PCR was performed on a Bio-Rad iQ5 Optical System Real Time PCR System (Bio-Rad, USA). Each reaction mixture was 20 μL containing 10 μL of SYBR Green PCR Master Mix (TaKaRa, Japan), 250 nM of each primer, and 6 μL of diluted first-strand cDNAs. The qRT-PCRs were run as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s in 96-well optical reaction plates. The Ct values were determined for three […]

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