title: 3' mRNA sequencing of piglet blood compartments
design: 3' mRNA sequencing of piglet blood compartments
library_name: RS1030_T3_06_s
library_construction_protocol: 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.  For whole blood RNA extractions, total RNA were extracted using the PAXgene ® Blood RNA Kit (QIAGEN), following the manufacturer instructions (manual protocol).  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.  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.  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). 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. 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. 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.