Computational protocol: Exploring the Genetic Resistance to Gastrointestinal Nematodes Infection in Goat Using RNA-Sequencing

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Protocol publication

[…] The data of RNA sequencing were received from the sequencing facility in FASTQ files that are exemplified tool command lines using a paired-end formatted file set []. Then we checked the overall sequence quality and the data with clean reads were obtained by trimming the adapter contaminants and filtering the low-quality reads. A quality control tool, Trimmomatic [], which can perform various useful forms of trimming was utilized for the quality control of our raw sequence data. [...] The reference genomes of Capra hircus (CHI_1.0) and the gene annotation information downloaded from the NCBI database (available at: ftp://ftp.ncbi.nlm.nih.gov/genomes/Capra_hircus/) were used in this analysis. After filtration by Trimmomatic, all clean reads of each sample were mapped to the reference sequence with TopHat-2 []. Subsequently, Cuffdiff were used to calculate gene and transcript expression in each sample following FPKM (fragments per kilobase of transcript per million mapped fragments) method to calculate gene expression []. In addition, to get an overview of gene expression difference between these two groups, a standardized reads-count of HTseq was fit to gplots to produce the heatmap.To gain insight into the function of the DEGs in the resistant and susceptible YWGs with the following parameters: Count = 2 and EASE = 0.01, DAVID (Database for Annotation, Visualization and Integrated Discovery) (available at: http://david.abcc.ncifcrf.gov/) [], bioinformatics resource tools were used for gene annotation and pathway analysis. Homo sapiens were used as a reference species due to the lack of data for Capra hircus in the DAVID website. The functions of genes in biological processes, cellular components, and molecular processes were annotated based on the GO categories. [...] The RNA sequencing data were validated through qRT-PCR, using a standard Prime SciptTM RT reagent Kit with gDNA eraser (Perfect Real Time), TAKARA Bio Inc. for cDNA production, and SYBR Green real time PCR master mix (Toyobo Co., Ltd., Osaka, Japan) for qRT-PCR reaction following manufacturers’ instructions. Twelve up- and down-regulated DEGs from were selected for qRT-PCR validation. The primers were designed from NCBI primer blast and primers were checked using OLIGO7 software (Molecular Biology Insights) in accordance with the sequences of the corresponding goat mRNAs in GenBank. A list of primers of selected genes for qRT-PCR experiment is shown in . Goat β-actin gene was used as a housekeeping gene in this qPCR validation experiments as a control gene for normalization of cDNA loading differences in this experiment. To avoid genomic DNA contamination, 2.0 µL of 5× gDNA eraser buffer, 1.0 µL of gDNA eraser, 1.0 µL of total RNA with a concentration of 1000 ng/µL, and 6.0 µl of RNase free dH2O were used to treat the RNA sample per reaction reagent. The reaction reagent of cDNA production contained 10.0 µL of gDNA elimination mix solution, 4.0 µL of 5× Prime Script buffer (for qRT-PCR), 1.0 µL of enzyme, 1.0 µL of RT primer mix, and 4.0 µL of RNase free dH2O. The qRT-PCR reaction contained 5.0 µL of SYBR Green real time PCR master mix (Toyobo Co., Ltd., Osaka, Japan), 0.2 µL of forward and reverse primer each, 1.0 µL of cDNA, and 3.6 µL of RNase free dH2O. The qRT-PCR reaction was performed on a Bio-Rad thermal cycler, CFX-384, real time system as follows: single cycle of denaturation at 95 °C for 5 min, 45 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 20 s, and extension at 72 °C for 5 s. […]

Pipeline specifications

Software tools Trimmomatic, TopHat, Cufflinks, HTSeq, gplots, DAVID, Primer-BLAST
Applications RNA-seq analysis, qPCR
Organisms Capra hircus, Caenorhabditis elegans
Diseases Gastrointestinal Diseases, Infection