Blood rna tubes

At the weekly blood draws, approximately 2.5 mL of whole blood was collected into K₂EDTA vacutainer tubes (BD Biosciences, Cat#367844), 2.0 mL into lithium heparin vacutainer tubes (BD Biosciences, Cat#367880, Franklin Lakes, NJ, USA), and 2.5 mL into blood RNA tubes (BD, PAXgene^®, Cat#762165, Franklin Lakes, NJ, USA). In addition, using 2 drops of whole blood per piglet, 2 slide smears were collected. Plasma samples were obtained by spinning the lithium heparin tubes at 1500× g for 10 min at 4 °C. A total of 500 uL of plasma, K₂EDTA tubes, and the blood smear samples were submitted to the Veterinary Medical Diagnostic Laboratory at Texas A&M University (College Station, TX, USA) for metabolic profiling, mineral panel, and complete blood count (CBC), respectively. Additionally, plasma samples were submitted to the Eurofins Craft Technologies Laboratory (Wilson, NC, USA) for plasma insulin measurements using a quantitative porcine insulin ELISA assay. At day 21, the blood (~45 mL) was collected into serum vacutainer tubes (BD Biosciences, Cat#368045, Franklin Lakes, NJ, USA) as well as into blood RNA tubes for further analysis. Serum tubes were incubated at room temperature for approximately 20 min and then centrifuged at 1500× g for 10 min at 4 °C. Serum aliquots were stored at −80 °C until further processing.

Total RNA from whole blood was isolated using Blood RNA tubes (BD) for blood collection. RNA integrity was assessed with a Bioanalyzer (Agilent Technologies). The TruSeq mRNA stranded kit from Illumina was used for the library preparation with 400 ng of total RNA as input. Library molarity and quality were assessed with the Qubit and Tapestation using a DNA High sensitivity chip (Agilent Technologies). Libraries were pooled at 2 nM and loaded for clustering on several lanes of a Single-read Illumina Flow cell to reach an average of 30 Million of reads per library. Reads of 100 bases were generated using the TruSeq SBS chemistry on an Illumina HiSeq 4000 sequencer.