Computational protocol: The association of hormone signalling genes, transcription and changes in shoot anatomy during moso bamboo growth

Similar protocols

Protocol publication

[…] The raw RNA‐seq in FASTQ format with a quality score of Phred ≥20 was indexed, trimmed and aligned. The clean reads were then aligned to the moso bamboo genome and gene set obtained from the National Center for Genome Research (http://www.ncgr.ac.cn/bamboo) using TopHat. Cufflinks was used to measure the relative abundance of transcripts with the FPKM method (fragments per feature kilobase per million reads; Ali et al., ). The identification of significant DGE models between different samples was performed with Cuffdiff (Trapnell et al., ). Differentially expressed transcripts (FDR value ≤0.05 and ≥1 fold change) were annotated and categorized automatically with Blast2go GO (Gene Ontology, http://www.blast2go.com/b2ghome.) To analyse phytohormone interactions, a network of all differentially expressed phytohormone‐related genes was constructed using WGCNA and visualized using Cytoscape (v.3.1.0). [...] To estimate the validity of the transcriptome sequencing, six genes were selected randomly and analysed with qRT‐PCR. qRT‐PCR of shoots at different growth stages was performed separately using culms after leaf expansion as the control. Total RNA was extracted using Trizol (Invitrogen), 2 mg of total RNA was reverse‐transcribed to the first‐stand cDNA using M‐MLVRT (Promega). The primers were designed using Primer 3 software (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) (Table ). TIP41 (tonoplast intrinsic protein 41) was used as an internal control (Fan et al., ; Peng et al., ). PCR was conducted using a fluorescent intercalating dye in a Light Cycler 480 SYBR Green I Master Mix (Roche, Mannheim, Germany; Roche). Cycling conditions were 95 °C for the initial 10 min, followed by 40 cycles of (10 s at 95 °C, 10 s at 60 °C and 20 s at 72 °C). All reactions were performed in triplicate, both technical and biological. […]

Pipeline specifications

Software tools TopHat, Cufflinks, Blast2GO, Primer3
Applications RNA-seq analysis, qPCR
Organisms Phyllostachys edulis