PMC:4502369 / 5722-15778 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4502369","sourcedb":"PMC","sourceid":"4502369","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4502369","text":"Materials and Methods\n\nAnimal material\nTissue samples of the testis and oviduct from Finnish Large White pigs were collected at slaughter. The dataset contains samples of ISTS-affected and control animals. Previously, we have studied the effect of ISTS on gene expression within the ISTS-associated region (Sironen et al. 2006, 2007, 2014), and we have shown that only the expression of SPEF2 and PRLR are affected. At slaughter, sows were approximately 4.5 months old and had not been bred. Boars were mature and had been used for breeding. The oviduct and testis tissue samples were collected in RNAlater buffer (Qiagen) and stored at −80°. For RNAseq analysis, four samples were used: the oviduct from an ISTS homozygous sow and a control sow, and the testis from an ISTS homozygous boar and a control boar. For RT-PCR, additional samples of two ISTS and control sows and boars were collected.\n\nRNA extraction and library preparation\nTotal RNA was extracted using RNeasy Midi kit (Qiagen) following the manufacturer’s instructions. The quality and concentrations of the RNA were checked using the Agilent 2100 Bioanalyzer (Agilent) and Nanodrop ND-2000 spectrophotometer (Thermo Scientific). Ribosomal RNA was removed with RibominusTM Eukaryote Kit for RNAseq (Invitrogen), and ribosomal RNA-depleted total RNA was fragmented using RNaseIII, to convert the whole transcriptome sample to RNA of a size appropriate for SOLiD System sequencing. After clean-up using the PurelinkTM RNA Micro kit, fragmented RNA samples with sufficient yield and an appropriate size distribution were ready for preparation of amplified cDNA libraries. Quality of the fragmentation was checked with the Bioanalyzer. The fragmented RNA sample was hybridized and ligated with the Adaptor Mix. RNA population with ligated adaptors was reverse-transcribed to generate single-stranded cDNA copies of the fragmented RNA molecules. After a clean-up step using the MinElute PCR Purification Kit, the sample was subjected to denaturing gel electrophoresis, and gel slices containing cDNA of the desired size range were excised. The size-selected cDNA was amplified using 15 cycles of PCR that takes place in the gel slices. This step appends required terminal sequences to each molecule and generates a sufficient template for SOLiD sequencing. After PCR, the amplified cDNA was cleaned using the PureLinkTM PCR purification kit. Libraries were quantitated with two different methods: Qubit fluorometer (Invitrogen) and quantitative PCR to ensure accuracy. The SOLiD Library TaqMan Quantitation Kit was used for determining the molar concentration of amplified template in a SOLiD library. In qPCR, the standard and unknown library template are amplified using two sequence-specific primers with a TaqMan fluorogenic probe labeled with FAM dye and a dye quencher. Uniformity of fragment size of libraries was confirmed with the Bioanalyzer. Templated bead preparation was performed by emulsion PCR (ePCR). SOLiD EZ Bead instrumentation was used for templated bead preparation.\n\nTranscriptomic data analysis\nThe colorspace reads obtained from the SOLiD sequencer were aligned against the pig reference genome (Sus scrofa build 10.2) using the standard whole transcriptome pipeline and the colorspace alignment tool provided by Applied Biosystems and supplied with the instrument (LifeScope v2.1). Reads associated with ribosomal RNA, transfer RNA, repeats, and other uninformative reads were filtered out during the process, as were reads with more than 10 potential alignments. For mapped reads, two mismatches per split were allowed, with two valid adjacent mismatches, which are likely to be SNPs, and were counted only as a single mismatch. After alignment to the reference genome, low-mapping-quality reads were discarded [mapQV (\u003c10)] and unique reads were associated with known genes based on UCSC annotations, and the number of reads aligned within each gene was counted. FPKM values were calculated following normalization of the data to remove variation between samples caused by nonbiological reasons, including library size and gene length using the Cufflinks software (v2.0.2) (Trapnell et al. 2012). Thus, the values generated are independent of the total number of reads in each sample and make the data comparable across the sample set. For differential expression analysis with the Cufflinks software, an FDR of no more than 0.01 and a minimum log fold-change of two was set as a threshold for a gene to be considered differentially expressed (DE) between groups.\nGene classification was performed with the Panther (Protein ANalysis THrough Evolutionary Relationships) classification system (Mi et al. 2005; Thomas et al. 2003). GO enrichment analyses of the Cufflinks results were performed using AgriGO (Du et al. 2010) and GOrilla (Eden et al. 2009). AgriGO accepted only protein IDs, which limited the power of the analysis due to a low number of identified genes with the correct ID. Thus, the knowledge of human gene function was exploited for the identification of enriched biological processes and classification analysis. For human genes, the list of all Ensembl annotated genes was used as the reference list. Associated gene names were obtained for pig genes by Biomart.\n\nGene variant detection\nFor identification of polymorphisms in the Finnish Large White transcriptome, we used the Genome Analysis Toolkit (GATK) (Depristo et al. 2011; McKenna et al. 2010; van der Auwera et al. 2013) program and SNP and Variation Suite v7 (Golden Helix, Inc., Bozeman, MT; www.goldenhelix.com) for filtering and annotation of the identified variants. The data were filtered based on the read depth (\u003e5), genotype quality (\u003e20), and quality score (\u003e80). The possible effect of amino acid substitutions on protein function was analyzed using SIFT (http://www.ensembl.org/Tools/VEP), and the effect on the secondary structure of the protein was analyzed using the CFSSP (http://www.biogem.org/tool/chou-fasman/) prediction tool.\n\nGene ortholog detection\nNovel transcripts detected by the Cufflinks suite were used to identify genetically active, but so far unannotated, regions in the Sus Scrofa genome. Detected sequences that were longer than 130 bp and that had a FPKM value of at least 5 for at least one of the samples were blasted against full genomes in the NCBI database “chromosomes” using the in-house R-package hoardeR (http://cran.r-project.org/web/packages/hoardeR/index.html). Hits that had an identity ratio larger than 0.9 were processed further. Due to the large amount of hits, subsequent analysis focused on hits in the top three species (bos taurus, homo sapiens, and ovis aries). The genome assemblies of the top species used were as follows: Homo sapiens, CRCh38; Bos Taurus, UMD3.1; and Ovis Aries, OAR3.1. Hits were then cross-checked against the gene annotations of the species so that the hits could be grouped into intergenic, intronic, and exonic hits. The R-package edgeR (Robinson, McCarthy and Smyth 2010) function exactTest was then applied for differential expression testing between the oviduct and testis samples and intronic/exonic hits. Values of FDR less than 0.01 were considered to be differentially expressed.\nGenomic regions with multiple hits within single genes were further investigated using a pairwise comparison approach based on sliding windows. Setting the window size to the smallest hit length, the genomic sequences of Sus Scrofa and the hit organism were divided into chunks of this window size, followed by a pairwise sequence similarity comparison between all chunks. The pairs with the largest similarities were reported and, for visualization of the region, indicated as a colored bar using a color spectrum between red and green for the degree of similarity. Pairs with low similarity (\u003c0.3) were omitted from the corresponding visualizations.\n\nRT-PCR and sequencing\nFor analysis of gene expression with RT-PCR, RNA of the testis and oviduct samples from two controls and ISTS homozygous animals were extracted (RNeasy Midi kit; Qiagen). Total RNA was reverse-transcribed with random primers and an RT-PCR kit (ImProm-II Reverse Transcription System; Promega) according to the manufacturer’s instructions. Synthesized cDNA was amplified using gene-specific primers (Supporting Information, Table S1). The housekeeping gene ribosomal S18 (RIBS18) was used as a reference gene to calculate the relative expression. cDNA samples were diluted to 20 ng/μl prior to use. The qPCR was performed with a ViiA 7 Real-Time PCR System in 96-well microtiter plates using Absolute qPCR SYBR Green ROX Mix (VWR). Amplification by qPCR contained 12.5 μl of Absolute qPCR SYBR Green Mix, 100 ng of cDNA, and 70 nM of each primer in a final volume of 25 μl. Amplifications were initiated with 15 min of enzyme activation at 95°, followed by 40 cycles of denaturation at 95° for 15 sec, primer annealing at 60° for 30 sec, and extension at 72° for 30 sec. All samples were amplified in triplicate, and the mean value was used in further calculations. Raw data were analyzed with the sequence detection software (Applied Biosystems) and relative quantitation was performed with GenEx software (MultiD). Ratios between the target and reference gene were calculated using the mean of these measurements. A standard curve for each primer pair was produced by serially diluting a control cDNA and used to correct for differences in amplification. A melting curve analysis was performed allowing single product-specific melting temperatures to be determined. No primer–dimer formations were generated during the application of 40 real-time PCR amplification cycles.\nFor sequencing, the RT-PCR amplicons were purified using ExoSAP-IT (Amersham Biosciences). PCR fragments were sequenced in both directions with the same primers used for amplification. Sequencing was performed on a MegaBace 500 capillary DNA sequencer (Amersham Biosciences) using DYEnamic ET Terminator Kits with Thermo Sequenase II DNA Polymerase (Amersham Biosciences).","divisions":[{"label":"title","span":{"begin":0,"end":21}},{"label":"sec","span":{"begin":23,"end":896}},{"label":"title","span":{"begin":23,"end":38}},{"label":"p","span":{"begin":39,"end":896}},{"label":"sec","span":{"begin":898,"end":3048}},{"label":"title","span":{"begin":898,"end":936}},{"label":"p","span":{"begin":937,"end":3048}},{"label":"sec","span":{"begin":3050,"end":5269}},{"label":"title","span":{"begin":3050,"end":3078}},{"label":"p","span":{"begin":3079,"end":4551}},{"label":"p","span":{"begin":4552,"end":5269}},{"label":"sec","span":{"begin":5271,"end":6012}},{"label":"title","span":{"begin":5271,"end":5293}},{"label":"p","span":{"begin":5294,"end":6012}},{"label":"sec","span":{"begin":6014,"end":7886}},{"label":"title","span":{"begin":6014,"end":6037}},{"label":"p","span":{"begin":6038,"end":7234}},{"label":"p","span":{"begin":7235,"end":7886}},{"label":"title","span":{"begin":7888,"end":7909}},{"label":"p","span":{"begin":7910,"end":9683}}],"tracks":[{"project":"2_test","denotations":[{"id":"25917919-16549801-43377632","span":{"begin":322,"end":326},"obj":"16549801"},{"id":"25917919-17610085-43377633","span":{"begin":328,"end":332},"obj":"17610085"},{"id":"25917919-24712415-43377634","span":{"begin":334,"end":338},"obj":"24712415"},{"id":"25917919-22383036-43377635","span":{"begin":4178,"end":4182},"obj":"22383036"},{"id":"25917919-15608197-43377636","span":{"begin":4690,"end":4694},"obj":"15608197"},{"id":"25917919-12952881-43377637","span":{"begin":4710,"end":4714},"obj":"12952881"},{"id":"25917919-20435677-43377638","span":{"begin":4804,"end":4808},"obj":"20435677"},{"id":"25917919-19192299-43377639","span":{"begin":4835,"end":4839},"obj":"19192299"},{"id":"25917919-21478889-43377640","span":{"begin":5432,"end":5436},"obj":"21478889"},{"id":"25917919-20644199-43377641","span":{"begin":5453,"end":5457},"obj":"20644199"},{"id":"25917919-19910308-43377642","span":{"begin":7015,"end":7019},"obj":"19910308"}],"attributes":[{"subj":"25917919-16549801-43377632","pred":"source","obj":"2_test"},{"subj":"25917919-17610085-43377633","pred":"source","obj":"2_test"},{"subj":"25917919-24712415-43377634","pred":"source","obj":"2_test"},{"subj":"25917919-22383036-43377635","pred":"source","obj":"2_test"},{"subj":"25917919-15608197-43377636","pred":"source","obj":"2_test"},{"subj":"25917919-12952881-43377637","pred":"source","obj":"2_test"},{"subj":"25917919-20435677-43377638","pred":"source","obj":"2_test"},{"subj":"25917919-19192299-43377639","pred":"source","obj":"2_test"},{"subj":"25917919-21478889-43377640","pred":"source","obj":"2_test"},{"subj":"25917919-20644199-43377641","pred":"source","obj":"2_test"},{"subj":"25917919-19910308-43377642","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93db","default":true}]}]}}