aggLDL_Insulin
Four individual experiments for each condition were submitted to the Gene Expression Center of the University of Wisconsin Biotechnology Center (UWBC) for total RNA extraction and library preparation. Samples were incubated with QIAzol Lysis Reagent (Qiagen, Hilden, Germany), follow of phase separation by centrifugation. After ethanol was added to the aqueous phase, samples were processed according to the RNAeasy protocol. The DNase treatment step was performed in the column, and RNA was eluted in nuclease-free water. Quantification and analysis of purity and integrity were performed for each sample using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent 2100 Bioanalzyer (Santa Clara, CA, USA), respectively. RNA samples presenting an optimal condition [RNA Integrity Number (RIN) > 8] were prepared according the TruSeq Stranded mRNA Sample Preparation Guide (Rev. E) using the Illumina TruSeq Stranded mRNA Sample Preparation Kit (Illumina Inc., San Diego, California, USA). For each library preparation, mRNA was isolated from 1000 ng of total RNA using poly-T oligo-attached magnetic beads. Later on, each poly-A-enriched sample was treated with divalent cations at elevated temperature for fragmentation. The obtained fragments were converted to double-stranded cDNA using SuperScript II (Invitrogen, Carlsbad, California, USA), RNaseH, DNA Pol I, and random primers.  The cDNA obtained was purified using AMPure XP beads (Agencourt, Beckman Coulter) following of Klenow DNA polymerase incubation to add an adenine base to the 3end of the blunt DNA fragments. After that, those fragments were ligated to unique dual index (UDI) adapters (IDT for Illumina- TruSeq RNA UD Index- catalog 20022371, IDT for Illumina - Nextera DNA Unique Dual Indexes, Set A and custom synthesized UDIs), which have a single thymine base overhang at their 3end. The adapter-ligated DNA products were purified with AMPure XP beads and amplified in a linker-mediated PCR reaction (LM-PCR) for 10 cycles using Phusion TM DNA Polymerase and Illuminas PE genomic DNA primer set followed by purification with AMPure XP beads. Finally, the quality and quantity of completed libraries were evaluated with Agilent DNA1000 chip (Agilent Technologies, Inc., Santa Clara, CA, USA) and Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA), respectively. Libraries were standardized to 2 nM and Paired-end 2  150 bp sequencing was performed using standard SBS chemistry on an Illumina NovaSeq6000 sequencer. The images obtained were analyzed using the standard Illumina Pipeline, version 1.8.2.