LIPOPROTEINS_Fractions_Healthy_Plasma_UG3

Libraries of highly purified exRNA carriers from human blood plasma pool Plasma samples were from 12 healthy subjects, 10 male and 2 pregnant female subjects. 500 μL of plasma was adjusted to a density of 1.006 g/mL by underlaying it with a 1.0035 g/mL potassium bromide (KBr) solution in a 4.7 mL OptiSeal tube. The sample underwent ultracentrifugation at 110,000 RPM for 4 hours using a fixed-angle TLA-110 rotor. After the first ultracentrifugation, the top 4 mL (triglyceride-rich lipoprotein fraction) was collected. The remaining 700 μL was mixed with 1.1 mL of 1.34 g/mL KBr solution to reach a final density of 1.21 g/mL, resulting in a total volume of 1.8 mL. This mixture was underlaid with 2.9 mL of 1.21 g/mL KBr solution in a new OptiSeal tube and ultracentrifuged at 58,000 RPM for 12 hours. Post-centrifugation, the top 2 mL was collected, concentrated to 250 μL using a 50 kDa Amicon filter, and injected into a high-performance liquid chromatography (HPLC) system equipped with a size-exclusion column. This step separated intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and various HDL subclasses. The bottom 2.7 mL from the second centrifugation was combined with 1.2 mL of 1.34 g/mL KBr solution to achieve a final density of 1.25 g/mL in a total volume of 3.9 mL. This solution was underlaid with 0.8 mL of 1.25 g/mL KBr solution in an OptiSeal tube and ultracentrifuged again at 110,000 RPM for 6 hours. The top 2 mL was then collected, concentrated to 250 μL, and subjected to further size-based separation using the same HPLC system. Fractions were collected using a temperature-controlled fraction collector, concentrated to 100 μL with a 50 kDa Amicon filter, and stored in 2% sucrose solution in -80C.RNA was extracted using Trisol LS and spin-column method. 8ul of isolated RNA were used to prepare libraries using BioliqX small RNA library kit. Libraries were sequinced using Illumina Nextseq 500 12 individual samples were sequenced in 4 lanes.