{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Yannick LIPPI"],"organism":["Sus scrofa"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16252"],"description":["This study describes the transcriptome of distinct blood compartments derived from whole blood collected from piglets. The dataset includes RNA-seq data from 20 samples representing five blood compartments: whole blood, serum, plasma, serum-derived exosomes, and plasma-derived exosomes. From each whole blood sample, the different fractions were carefully prepared using optimized and compartment-specific protocols. RNA-seq libraries were prepared using the LEXOGEN QuantSeq 3′ mRNA-Seq V2 Library Prep Kit with protocol adaptations for whole blood, plasma, serum, and exosome samples. Sequencing was performed on the Illumina NextSeq2000 platform in SR75. The 20 raw demultiplexed FASTQ files are part of this accession. Bioinformatic pre-processing was conducted using the Nextflow nf-core/rnaseq pipeline to ensure high sequence quality and robust transcriptome assembly and quantification. Each file contains raw sequencing reads along with base quality scores. Bioinformatic and statistical analyses explored the variability in gene detection and gene expression variability according to the five blood compartments."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Nucleic Acid Extraction - Plasma and serum RNA extractions were performed using the Plasma/Serum RNA Purification Kit (NORGEN) following the manufacturer's instructions, using 200 µL aliquots of plasma or serum.","Nucleic Acid Extraction - Exosome purification was performed on 1 ml aliquots of plasma or serum using Plasma/Serum Exosome Purification Kit (NORGEN), following the manufacturer's instructions. Then, all 200 µL of the previously obtained exosomes were used to extract exosomal RNA using the Exosomal RNA Isolation Kit (NORGEN), following the manufacturer's instructions.","Library Construction - For serum and plasma samples, RNA-Seq libraries were generated from 5µl of raw total RNA extracts, following the manufacturer’s low input/low quality protocol version. First-strand cDNA synthesis was performed using oligo-dT priming, and the standard RNA Removal Solution was applied prior to second-strand synthesis initiated by random priming. Double-stranded cDNA fragments were indexed with UDI and amplified with twenty-one PCR cycles.","Nucleic Acid Extraction - For whole blood RNA extractions, total RNA were extracted using the PAXgene ® Blood RNA Kit (QIAGEN), following the manufacturer instructions (manual protocol).","Sample Collection - Samples were collected from four 5-week-old crossbred female piglets (Duroc DanBred X Hybride DanBred). Animal experimentation procedures were approved by the Ethics Committee of Pharmacology-Toxicology Occitanie-Toulouse) (Toxcom 310 LSV), in accordance with the European Directive on the protection of animals used for scientific purposes (2010/63/EU). The animals were acclimatized for one week in the animal facility, then euthanized by electronarcosis before exsanguination, during which blood samples were collected. Two animals were sampled in October 2023 (animals 2 and 6) and two others in April 2024 (animals 3 and 4). Depending on the type of fluid to be studied, blood was collected from each animal in three types of tubes : - approximately 2.5 ml of blood in a PAXgene® RNA Blood tube (QIAGEN): 1 tube per animal; for total RNA extraction from whole blood - approximately 4 ml of blood in EDTA (anticoagulant) tubes : 4 to 6 tubes per animal ; for RNA extraction from plasma. - approximately 4 ml of blood in dry tubes with a coagulation activator: 4 to 6 tubes per animal ; for RNA extraction from serum. Each tube was homogenized 10 times by inversion. PAXgene® tubes were left at room temperature for two hours, then frozen at -20°C overnight before being stored at -80°C until extraction. EDTA tubes were placed in melting ice until centrifugation at 2000 g for 10 minutes at 4 °C (within two hours of collection). Dry tubes were left at room temperature for 30 minutes before centrifugation at 1300 g for 10 minutes at room temperature. For each animal, the plasma or serum collected from the different tubes are pooled, homogenized, and then divided into aliquotes.","Sequencing - The 20 libraries were multiplexed at equimolar concentrations and transferred to the INRAE PGTB sequencing facility (doi :10.15454/1.5572396583599417E12). the pool was loaded into a P2 Flow Cell using NextSeq 1000/2000 XLEAP-SBS P2 Reagent kit (100 Cycles), with 10% of PhiX spike-in to compensate for low diversity expected in serum/plasma samples. Sequencing was performed using single-end 76 bp reads on the Illumina NextSeq 2000 Sequencing System (Illumina, USA).","Library Construction - For exosomes extracts samples derived from serum or plasma, RNA-Seq libraries were generated from 5µl of raw total RNA extracts, following the manufacturer’s low input/low quality protocol version. First-strand cDNA synthesis was performed using oligo-dT priming, and the standard RNA Removal Solution was applied prior to second-strand synthesis initiated by random priming. Double-stranded cDNA fragments were indexed with UDI and amplified with twenty-six PCR cycles.","Library Construction - RNA libraries were prepared with QuantSeq 3’ mRNA-seq V2 Library Prep Kit FWD with 12-nt Unique Dual Indices (UDI) for Illumina (Lexogen, Austria).","Library Construction - For whole blood samples, RNA-Seq libraries were generated from 200 ng of total RNA, following the manufacturer’s standard input protocol and with the RNA Removal Solution Globin Block, Sus scrofa (RS-GBSs, Cat. No. 071). The first-strand cDNA was synthesized by reverse transcription with oligo-dT priming. Prior to second-strand synthesis, RS-GBs containing porcine globin-specific oligonucleotide mixtures were used to selectively bind globin mRNA-derived cDNA strands and prevent their extension during second-strand synthesis initiated by random priming. Double-stranded cDNA fragments were indexed with single indices and amplified with seventeen PCR cycles."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Sequence Alignment - The bioinformatic analysis pipeline was executed with Nextflow v23.10.0 (Di Tommaso et al., 2017) and processed using nf-core/rnaseq v3.16.0 (https://doi.org/10.5281/zenodo.10471647 ) of the nf-core collection of workflows (Ewels et al., 2020), utilising reproducible software environments from the Bioconda (Grüning et al., 2018) and Biocontainers (da Veiga Leprevost et al., 2017) projects. Extra parameters were adapted to 3' RNA-seq reads: --extra_star_align_args '--alignIntronMax 1000000 --alignIntronMin 20 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --outFilterMismatchNmax 999 --outFilterMultimapNmax 20 --outFilterType BySJout --outFilterMismatchNoverLmax 0.1 --clip3pAdapterSeq AAAAAAAA'. Reads were aligned to pig genome reference Sscrofa11.1 (build GCA_000003025.6) and the quantification of reads was resumed to genes defined in Ensembl release 112 annotation. the count table is provided as 'processed data file'.","Data Transformation - Biostatistics treatments were performed under R v4.3.0 (R Core Team, 2023), edgeR package (Robinson et al., 2010) and Bioconductor packages (Huber et al. 2015). Briefly, raw count table was filtered to eliminate undetected genes using filterByExpr function with min.count=10 parameters. Normalization factors were calculated using TMM method. Then the common, trended and tagwise negative binomial dispersions were estimated with the robust mode of estimateDisp function. Normalized log2 CPM (counts per million reads) were exported to generate the normalized data file which is provided as 'processed data file'."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["NextSeq 2000"],"study_type":["RNA-seq of total RNA"],"species":["Sus scrofa"],"pubmed_authors":["Yannick LIPPI"],"additional_accession":[]},"is_claimable":false,"name":"3' mRNA sequencing of piglet blood compartments","description":"This study describes the transcriptome of distinct blood compartments derived from whole blood collected from piglets. The dataset includes RNA-seq data from 20 samples representing five blood compartments: whole blood, serum, plasma, serum-derived exosomes, and plasma-derived exosomes. From each whole blood sample, the different fractions were carefully prepared using optimized and compartment-specific protocols. RNA-seq libraries were prepared using the LEXOGEN QuantSeq 3′ mRNA-Seq V2 Library Prep Kit with protocol adaptations for whole blood, plasma, serum, and exosome samples. Sequencing was performed on the Illumina NextSeq2000 platform in SR75. The 20 raw demultiplexed FASTQ files are part of this accession. Bioinformatic pre-processing was conducted using the Nextflow nf-core/rnaseq pipeline to ensure high sequence quality and robust transcriptome assembly and quantification. Each file contains raw sequencing reads along with base quality scores. Bioinformatic and statistical analyses explored the variability in gene detection and gene expression variability according to the five blood compartments.","dates":{"release":"2025-12-16T00:00:00Z","modification":"2026-05-30T15:35:20.47Z","creation":"2025-11-22T21:37:28.952Z"},"accession":"E-MTAB-16252","cross_references":{"ENA":["ERP185508"],"EFO":["EFO_0002944","EFO_0004170","EFO_0009653","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0004184"]}}