GSM8626807: PIG21-ALAC-Jej; Sus scrofa; RNA-Seq
Tissues were collected immediately after euthanasia, follow by snap-freezing in liquid nitrogen, and stored at -80°C until further analysis. Tissues were manually ground in liquid nitrogen using a mortar and pestle. 25-30 mg of cryoground tissue was extracted using a modified Folch extraction, and RNA was extracted from the resulting cell layer (All Prep DNA/RNA/Protein Kit, Qiagen, Germantown, MD). RNA samples were quantified using the Qubit RNA HS Assay Kit and Quibit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA), and sample quality was assessed using a Lab Chip GX Touch HT (Perken Elmer, Waltham, MA). Isolated RNA was submitted to the UC Davis DNA Technologies and Expression Analysis Core to generate 3' Tag-Seq libraries with unique molecular indices (UMIs). To generate Illumina-compatible multiplexed sequencing libraries, barcoded 3'Tag-Seq libraries were prepared using the QuantSeq FWD kit (Lexogen, Vienna, Austria) according to the manufacturer's recommendations.  The fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis via Bioanalyzer 2100 (Agilent, Santa Clara, CA).  The libraries were quantified fluorometrically on a Qubit instrument (LifeTechnologies, Carlsbad, CA), and pooled in equimolar ratios. 232 libraries were sequenced on 2.5 lanes of NextSeq500 sequencer.