GSM2947962: Urine_WT_3; Mus musculus; ncRNA-Seq
Plasma, basal bile, urine, and livers were collected from wild-type (WT) and SR-BI-deficient (B6;129S2-Scarb1tm1Kri/J, SR-BI KO) mice. Mice were anesthetized with urethane (1g/kg, i.p.). The common bile duct was ligated and the gall bladder cannulated to divert bile into collection tubes. Basal bile was collected for a period of 30 min. Mice were then exsanguinated, blood was collected from the abdominal aorta in EDTA coated tubes and placed on wet ice, and tissues were dissected and snap frozen in liquid nitrogen. Plasma and tissues were stored at -80oC prior to analysis.  All animal procedures were completed under active and approved IACUC protocols. To separate HDL and apolipoprotein B (APOB)-containing lipoproteins from mouse plasma, 200 µL of 0.22-µm filtered-plasma samples were diluted to 500 µL in size-exclusion chromatography (SEC) running buffer (10 mM Tris-HCl, 0.15 M NaCl, 0.2% NaN3) and injected an ÄKTA SEC system (GE Healthcare) with three in-series Superdex-200 Increase gel filtration columns (10/300 GL; GE Healthcare). Samples were applied to the column with a flow rate of 0.3 mL/min at room temperature and eluate collected as 72 x 1.5 mL fractions using a F9-C 96-well plate fraction collector (GE Healthcare). Each fraction was analyzed for total protein (BCA; Pierce), total cholesterol (Raichem), and triglycerides (Raichem) to identify fractions corresponding with HDL and APOB particles. Due to the SEC set-up, we were not able to separate VLDL from LDL particles, and thus, we collected fractions covering both lipoprotein classes, referred to here as APOB. Fractions corresponding with each lipoprotein group were pooled, concentrated with Amicon Ultra-4 10 kDa centrifugal filters (Millipore) to <200 µL volume, and protein concentrations were quantified by BCA assays (Pierce). Based on the distribution of total cholesterol, triglycerides, and protein, fractions corresponding to HDL and APOB were collected, pooled, and concentrated. To differentiate lipoprotein sRNA signatures from liver and biofluids, and determine the impact of SR-BI-deficiency, samples were collected from Scarb1-/- (SR-BI KO) and wild-type (WT) mice. Total RNA was extracted from HDL (WT N=7, SR-BI KO N=7) and APOB (WT N=7, SR-BI KO N=7) particles, as well as livers (WT N=7, SR-BI KO N=7), bile (WT N=7, SR-BI KO N=6), and urine (WT N=5, SR-BI KO N=6). RNA was isolated from equal inputs of either bile (volume), liver (mg), HDL (protein) or APOB (protein) using miRNAEasy Mini kits (Qiagen). Specifically, 30 µL of primary bile, 120 μg of APOB, 180 μg of HDL or 20 mg of liver were added to 1 mL of Qiazol. Livers were homogenized in Qiazol with High-Impact Zirconium beads using a Bead Bug Homogenizer (Benchmark Scientific).  After removal of beads, subsequent steps for liver RNA extraction were followed according to manufacturer's protocol. Bile, APOB and HDL RNA isolations were processed according to manufacturer's protocol, except that after addition of ethanol, samples were incubated at -80oC overnight before application to isolation columns, and were eluted with a volume of 50 μL. Liver RNA samples were quantified by Take3 plates (BioTek). NEXTflex Small RNA Library Preparation Kits v3 for Illumina® Platforms (BioO Scientific) were used to generate cDNA libraries for sRNA-seq. Briefly, 1 µg of liver total RNA was used as input for adapter ligation, as per manufacturer's protocol. For bile, APOB and HDL RNA, 10.5 μL (21%) of the RNA isolation eluate was used as input for adapter ligation. Library generation was performed according to manufacturer's protocol (BioO Scientific) with a modification to the amplification step, as liver libraries received 18 cycles and bile, APOB and HDL libraries received 25 cycles. After amplification, samples were size-selected using a Pippin-Prep (Sage Science) -- set for a range of 135-200 nts in length -- and subsequently purified and concentrated using DNA Clean and Concentrator 5 kit (Zymo). Individual libraries were then screened for quality by High-Sensitivity DNA chips using a 2100 Bioanalyzer (Agilent) and quantified using High-Sensitivity DNA assays with Qubit (Life Technologies). Equal concentrations of all individual libraries were pooled for multiplex sequencing runs, and concentrated using DNA Clean and Concentrator 5 kit (Zymo). For rigor in down-stream comparisons, all 66 sequencing libraries were randomized and run independently on three individual sequencing lanes. Single-end sequencing (75 cycles) of multiplexed libraries were performed on an Illumina NextSEQ 500 at the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core laboratory. Each library was sequenced at an average depth of 16.28 million reads/sample.
GEO Accession
GSM2947962