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| Status |
Public on Jun 01, 2019 |
| Title |
cajal2 |
| Sample type |
SRA |
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| Source name |
primary interstitial cells of Cajal
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| Organism |
Ovis aries |
| Characteristics |
tissue: Duodenum
age: about 8 months old
virus strain infection: none
hours of virus infection: 0
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| Treatment protocol |
Monolayers of ICC (approximately 80% confluence) infected with or without BVDV TC (100 TCID50/0.1 ml) for 36 h
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| Growth protocol |
The duodenum of Merino sheep (about 8 months old) used to isolate primary ICC was collected from Hualing Livestock Slaughterhouse in Urumqi, Xinjiang. The duodenum was washed with 0.1 M phosphate-buffered saline (PBS) plus 50 mg/ml of Penicillin/Streptomycin solution several times. The mucosa and submucosa of the duodenum were removed under an anatomic microscope, and strips of muscle were cut into small pieces (l–2 mm3) and digested with Hank’s balanced salt solution plus 1 mg/mL Collagenase (type II; Thermo Fisher Scientific, catalog number: 17101015) and trypsin (0.05%; Thermo Fisher Scientific, catalog number: 25200056) in a standard cell incubator for 30 min. To stop digestion, an equal volume of Medium 199 (Thermo Fisher Scientific) plus 20% FBS, 5% Penicillin/Streptomycin solution, 5 ng/mL of human stem cell factor (HEOPP-1902, Cyagen Biosciences, Guangzhou, China), and 1×MEM non-essential amino acids solution (Thermo Fisher Scientific, catalog number: 11140050) were used. The digested mixture was passed through a 200-mesh screen, and 20% Ficoll 400 (Sigma) density gradient centrifugation was performed to purify ICC as described previously (Zhang et al., 2017)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Monolayers of ICC infected with/without BVDV were harvested and lysed using TRIzol solution.
Sequencing libraries were generated using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, Beverly, MA, USA) following the manufacturer’s recommendations.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
In order to guarantee the data quality which was used to analysis, the useful Perl script was used to filter the original data (Raw Data). The steps are: (1) Trim Smart-seq2 public primer sequence from the Reads; (2) Remove the contaminated reads for adapters; (3) Remove the low quality reads; (4) Remove the reads whose N base more than 5% for total bases.
The reference genomes and the annotation file were downloaded from ENSEMBL database (http://www.ensembl.org/index.html). Bowtie2 v2.2.3 was used for building the genome index, and Clean Data was then aligned to the reference genome using HISAT2 v2.1.0.
The IGV (Integrative Genomics Viewer) was used to view the mapping result by the Heatmap, histogram, scatter plot or other stytle.
Reads Count for each gene in each sample was counted by HTSeq v0.6.0, and RPKM (Reads Per Kilobase Millon Mapped Reads) was then calculated to estimate the expression level of genes in each sample, the formula is shown as：RPKM=(106*R)/(NL/103). R is the number of reads in a certain sample that is assigned to a certain gene, N is the total number of mapped reads in the certain sample and L is the length of the certain gene.
DEGseq v1.18.0 was used for differential gene expression analysis between two samples with non biological replicates. Under the assumption that the number of reads derived from a gene (or transcript isoform) follows abinomial distribution, DEGseq is proposed based on MA-plot and widely used for differential gene expression analysis. The P-value could be assigned to each gene and adjusted by the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with q≤0.05 and |log2_ratio|≥1 are identified as differentially expressed genes (DEGs). DESeq2 v1.6.3 was designed for differential gene expression analysis between two samples with biological replicates under the theoretical basis obeys the hypothesis of negative binomial distribution for the value of count. Differing from DESeq, DESeq2 estimates the depth parameters of samples and gene-specific, as well as uses "linear regression" for dispersion to "shrink", which was mainly considering genes of the same expression level may share the similarity deviations or own their the expression characteristics. DESeq2 estimates the expression level of each gene in per sample by the linear regression, then calculates the p-value with Wald test. Finally the p-value was corrected by the BH method. Genes with q≤0.05 and |log2_ratio|≥1 are identified as differentially expressed genes (DEGs).
The GO (Gene Ontology, http://geneontology.org/) enrichment of DEGs was implemented by the hypergeometric test, in which p-value is calculated and adjusted as q-value, and data background is genes in the whole genome. GO terms with q<0.05 were considered to be significantly enriched. GO enrichment analysis could exhibits the biological functions of the DEGs. KEGG (Kyoto Encyclopedia of Genes and Genomes, http://www.kegg.jp/) is a database resource containing a collection of manually drawn pathway maps representing our knowledge on the molecular interaction and reaction networks. The KEGG enrichment of DEGs was implemented by the hypergeometric test, in which p-value was adjusted by multiple comparisons as q-value. KEGG terms with q<0.05 were considered to be significantly enriched.
Genome_build: Bos taurus
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample.
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| Submission date |
Nov 08, 2018 |
| Last update date |
Jun 01, 2019 |
| Contact name |
Qiang Fu |
| E-mail(s) |
fq198505@gmail.com
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| Phone |
869932058002
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| Organization name |
Shihezi University
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| Street address |
Bei 4 Road
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| City |
Shihezi |
| State/province |
Xinjiang |
| ZIP/Postal code |
832000 |
| Country |
China |
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| Platform ID |
GPL19778 |
| Series (1) |
| GSE122344 |
RNA-Seq-based transcriptome profiling of primary interstitial cells of Cajal derived from the small intestine of Merino sheep in response to bovine viral diarrhea virus infection |
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| Relations |
| BioSample |
SAMN10397161 |
| SRA |
SRX4997323 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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