Computational protocol: Unique genetic responses revealed in RNA-seq of the spleen of chickens stimulated with lipopolysaccharide and short-term heat

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Protocol publication

[…] Quality control of RNA-seq reads was conducted using FASTQC and FASTX Clipper (version 0.0.13; with the following options; phredd score of 30, minimum base pair length of 30, and adapter sequences were removed.Reads were mapped to the Gallus gallus genome version 4.0 (4.78 GTF Ensembl) using Tophat (version 2.0.9) [] using default parameters. Counting of mapped reads to a gene was done using HTSeq (version 3.0). includes the average (N = 4/treatment group) number of generated reads before and after quality filtering, number of mapped and the percentage of transcriptome coverage. There are 15,508 annotated coding genes in Gallus gallus 4.0 genome. To calculate transcriptome coverage, we used the average number of annotated genes that were expressed in our dataset and divided by the number of annotated coding genes. The resulting number is within range of predicted coverage based on sequencing depth []. Differential gene expression was detected using EdgeR (version 1.00), with Benjamini Hochberg method used for multiple testing correction, and maximum FDR ≤ 0.05. A pairwise comparison was used to detect DEG within breed by contrasting each treatment with the most naive group, i.e. TN_PBS. The contrasts contained 4 individuals per treatment group and were as follows; F_TN_PBS vs F_HS_PBS, F_TN_PBS vs F_TN_LPS, F_TN_PBS vs F_HS_LPS, B_TN_PBS vs B_HS_PBS, B_TN_PBS vs B_TN_LPS, and B_TN_PBS vs B_HS_LPS. [...] We used Fluidigm gene expression technology to confirm the mRNA expression detected by RNA-seq in the current study. As described in the Library Generation and Sequencing section, total RNA was isolated and treated with a DNA-free kit. Gene expression analysis was performed using a microfluidic Reverse Transcription quantitative PCR (RT-qPCR) (Fluidigm Corporation, San Francisco, CA, USA). All procedures were conducted according to manufacturer’s recommendations, unless otherwise noted. Briefly, 50 ng of the total RNA was reverse transcribed using the Fluidigm Reverse Transcription Master Mix (Fluidigm Corporation, San Francisco, CA, USA). cDNA was pre-amplified with PreAmp Master Mix (Fluidigm Corporation, San Francisco, CA, USA), using 12 cycles of pre-amplification. Exonuclease I (New England Biolabs, UK) treatment was applied to remove unincorporated primers. Pre-amplified and purified cDNA samples were diluted 10x in TE buffer and stored at -20°C until further analyses. RT-qPCR analysis was completed for 22 target genes and 2 reference genes, listed in . These genes were selected to assay because they represented a range of fold change expression values from extremely changed (LFC ≥ ±30) to not changed due to treatment (LFC = 0). The FlexSix Integrated Fluid Circuits (IFCs) (Fluidigm Corporation, San Francisco, CA, USA) was used to assay mRNA expression. Sample assay included 1.35 μl of pre-amplified and Exo I treated cDNA, 1.5 μl of the SsoFast™ EvaGreen®Supermix with Low ROX™ (2x) (Bio-Rad) and 0.15 μl of the FlexSix Delta Gene Sample Reagent (Fluidigm Corporation, San Francisco, CA, USA). Primer assays were prepared as 20 μl stock by mixing 1μl of each primer (100 μM) with 10 μl of the 2x Assay Loading Reagent and adjusted to 20 μl with DNA suspension buffer (low EDTA TE buffer). The samples, assays and the loading reagents were then loaded onto IFCs microfluidic channels using the RX loading station (Fluidigm Corporation, San Francisco, CA, USA). RT-qPCR was performed on the Biomark™ HD (Fluidigm Corporation, San Francisco, CA, USA) using the fast program that consisted of an incubation step at 95°C for 60 s followed by 30 cycles: 96°C for 5s and 60°C for 20s. Fluorescence emission was recorded after each cycling step. Upon RT-qPCR completion, melting curves were generated by increasing temperature from 60 to 95°C, with continuous fluorescence acquisition.RT-qPCR data were analyzed as follows: raw qPCR data were analyzed and checked for quality using Real-Time PCR Analysis Software (Fluidigm Corporation, San Francisco, CA, USA). Main effects of the stimulation of the spleen were estimated using least square means method implemented in JMP Pro 10.0.2 software (SAS Institute, Cary, NC, USA). Chicken line (Fayoumi or broiler), thermal treatment (TN or HS), and immune stimulus (PBS and LPS) as well as the interaction between line and treatments were fitted in the model. Analyses were performed separately for each line, gene, and treatment using dCt values (Ct target–Ct reference). To determine the relative gene expression, ddCt method was used []. Delta Ct values were obtained by normalizing the Ct values of the target genes with the geometrical mean of the two reference genes (H6PD and RPL4). Fold induction of the gene expression was estimated as 2-ddCt. Untreated (control) samples were used as calibrators. […]

Pipeline specifications

Software tools FastQC, FASTX-Toolkit, TopHat, HTSeq, edgeR, JMP Pro
Applications Miscellaneous, RNA-seq analysis
Organisms Gallus gallus
Diseases Chemical and Drug Induced Liver Injury