title: GSM7113868: Pig 4, Myocardial infarction and HPAC treatment_RZ; Sus scrofa domesticus; RNA-Seq
library_construction_protocol: Following excision of LV core from IZ, BZ, and RZ, samples were rinsed in 1X PBS (10x PBS Solution, 1.37M NaCl, 0.027M KCl and 0.119M phosphates, pH 7.4, Fisher BioReagents, Ref: BP3994) to remove any residual blood. Tissue was submerged in RNAlater® (Sigma-Aldrich, St. Louis, MO, Ref: R0901-500ML) and placed in 4ºC for 48 hours prior to storing in -80ºC.   The epicardial surface of the LV core biopsy (1/3 of the tissue) was utilized for RNA extraction. Tissue samples were submerged in 400μL of Trizol reagent (ThermoFisher, Carlsbad, CA, Ref:15596026) and ground for 30 seconds in a 2mL flat centrifuge tube using an OMNI THQ digital rotor-stator (OMNI, Kennesaw, GA, Ref:12-500) at 15,000RPM and hard tissue OMNI TipTM (OMNI, Kennesaw, GA, Ref: 30750H). Each sample was ground using a fresh tip to prevent cross contamination. Tubes were gently rocked back and forth to mix contents and were left undisturbed at room temperature for 10 minutes. Thereafter, 100μL of chloroform (Millipore-Sigma, Milwaukee, WI, Ref:288306) was added, mixed, and centrifuged (Eppendorf 5424R, Hamburg, Germany) at 15,000RPM for 5 minutes. The supernatant was transferred to a new tube, to which lysis buffer from the Qiagen RNEasy Plus Mini kit (Qiagen, Hilden, Germany, Ref: 74134) containing BME was added to bring samples up to a volume of 650μL. The total volume of sample was then transferred to a gDNA eliminator column from the Qiagen RNEasy Plus Mini kit and centrifuged in the Eppendorf at 8,000RPM for 30 seconds. The flow-through from the centrifugation step was then mixed in a 1:1 volume ratio of 70% EtOH, resulting in a final concentration of 35% EtOH by volume of the total sample volume. The subsequent steps were followed according to kit manufacturer's protocol.    RNA samples were assessed for quality with an Advanced Analytics Fragment Analyzer (High Sensitivity RNA Analysis Kit – Ref: DNF-471-0500) and quantity with a Qubit RNA quantification kit (Qubit® RNA HS Assay KitAssay Kit – Ref: Q32852). RNA Quality Number (RQN) of samples ranged from 5.2 to 9.1. Given satisfactory quality and quantity, samples were used for library builds. A total amount of 1μg RNA per sample was used as input material for the RNA sample preparations. Libraries were constructed using NEBNext®UltraTMRNA Library Prep Kit for Illumina® (NEB, USA) according to manufacturer's recommendations. Index codes were added to attribute sequences to each sample. Poly-T oligo-attached magnetic beads were used to purify mRNA from the total RNA sample, and fragmentation was achieved using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). Subsequently, second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. To prepare for hybridization, NEBNext Adaptor with hairpin loop structure were ligated following 3' end adenylation. The AMPure XP system (Beckman Coulter, Beverly, USA) was used to isolate cDNA fragments of approximately 150-200 base pairs long. PCR was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer following addition of 3μl USER Enzyme (NEB, USA) with size-selected, adaptor-ligated cDNA at 37°C for 15 minutes followed by 5 minutes at 95°C. Lastly, PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system. A total amount of 1μg RNA per sample was used as input material for the RNA sample preparations. Libraries were constructed using NEBNext®UltraTMRNA Library Prep Kit for Illumina® (NEB, USA) according to manufacturer's recommendations