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Sample GSM2694724

Query DataSets for GSM2694724

Status

Public on Aug 15, 2019

Title

YCCZ5

Sample type

SRA

Source name

mycelium

Organism

Cordyceps militaris

Characteristics

strain: YCC
passage number: 5th passage
tissue: mycelium

Treatment protocol

Then 5 ml mixture were inoculated into rice-medium to culture the fruiting body. The rest mixture was vacuum filtrated for 1 hour to collect the mycelium and pulverized in liquid nitrogen immediately

Growth protocol

A clump of C. militarisYCC strain hyphae with strong vitality were picked up from slant culture-medium and added into 100 ml improved potato dextrose agar medium (Glucose 2.0 g，KH2PO40.3 g，MgSO40.15 g，Vitamin B2 1 mg，Peptone0.3 g，Yeast extract 0.5 g，Distilled water 100 ml) and cultured in the dark environment at 25±1℃, 130-140 rpm for 72 hours.

Extracted molecule

total RNA

Extraction protocol

Total RNA of mycelium was extracted using RNeasy Plant Mini kit and the residual genomic DNA was digested using RNase-free DNase I according to the manufacturer’s protocol.
Poly(A) mRNA was enriched by beads with oligo (dT) and interrupted by fragmentation buffer into suitableshort fragment.100 ng purified and enriched mRNA per sample were used to construct the respective cDNA libraries using NEBNext® UltraTM RNA Library Prep Kit for Illumina (NEB,USA). cDNA fragments of 200 bps (±25 bps) were selected and purified by gel-electrophoresis as the templatesfor amplification with PCRfor end reparation andadding poly (A). Six cDNA library would be accomplished after been connectedwith sequencing adaptors

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

cultured in the dark for 72 hours

Data processing

The raw data was obtained by Illumina Hiseq TM 2500 and the quality was assessed using the FastQC and Fast X-Tool kit to detect the base composition, base quality, sequencing saturation and randomness.
Clean reads were obtained by removing adaptor sequences, unknown sequences, low quality reads (Q≤5) and sequences with a length of less than 50 bp.
The clean reads were assembled by Cuffmerge software, then directly mapped to the ribosome RNAdatabase and genome of C. militaris [30] respectively using Bowtie aligner and TopHat2 aligner.
The filtered mapping data were used to reconstruct the transcripts by reference annotation based transcripts (RABT) method with Cufflinks software.
Gene identification and structure optimization
Alternative Splicing (AS) assay, Single-nucleotide polymorphism (SNP)/InDel and SNP mediated RNA editing assay
The expression levels of genes were calculated using FPKM (Fragments Per Kilobase of transcript per Million mapped reads) and the quality was assessed by FPKM distribution assay of different samples.
The gene expression levels were compared based on the DEGSeq and P values were used to adjust the significance of multiple comparisons using the Benjamini and Hochbergmethod. Differentialexpression genes (DEGs)were filtered and identified with the edgeR (http://www.Bioconductor.org/packages/release/bioc/html/edgeR.html) criteria as False Discovery Rate (FDR) and the thresholds was FDR≤0.001 and |log2 Fold Change (Log2FC)|≥1.Cluster analysis of all genes expression patterns from different samples was accomplished too. We also performed the correlation heat ma pand sample cluster analysis based on the DEGs.
gene ontology terms (GO) and Kyoto Encyclopedia of Genes andGenomes (KEGG) enrichment analyses
Genome_build: Cordyceps militaris CM01 Genome sequencing and assembly (https://www.ncbi.nlm.nih.gov/bioproject/41129)
Supplementary_files_format_and_content: All files are named with sample name followed by content name using files format of excel. The processed data include the Gene structure optimization, Genes expression, Pearson correlation coefficient, RNA editing events based on SNP and SNP-InDel of all six samples; the differentially expressed genes of YCCZ1-vs-YCCZ2, YCCZ1-vs-YCCZ3, YCCZ1-vs-YCCZ4, YCCZ1-vs-YCCZ5 and YCCZ1-vs-YCCZ6; alternative splicing of of YCCZ1-vs-YCCZ2, YCCZ1-vs-YCCZ3, YCCZ1-vs-YCCZ4, YCCZ1-vs-YCCZ5 and YCCZ1-vs-YCCZ6.

Submission date

Jul 05, 2017

Last update date

Aug 15, 2019

Contact name

Juan YIN

E-mail(s)

13275170020@163.com

Phone

17788351530

Organization name

Jiangsu University of Science and Technology