GSM4970413: A16106_Thalamus(N512); Macaca fascicularis; RNA-Seq
Between 30-50 mg of amygdala, hippocampus, or thalamus of non-human primate tissues were added to 1mL TRIzol with 5mm stainless steel beads (Qiagen, Germantown, MD). Tissues were homogenized using Qiagen's TissueLyser LT for 2 mins at 40Hz. Homogenized tissues were centrifuged at 10,000 RPM for 2 mins. The supernatant was transferred to a new tube and an equal volume of 100% ethanol was added. Total RNAs, including small and micro RNAs, were extracted using Direct-zol RNA Miniprep Plus (Zymo Research, Irvine, CA) according to manufacturer's protocol. Extracted RNAs were quantified on a Qubit 4 Fluorometer using the RNA HS Assay Kit (Thermo Fisher Scientific, Grand Island, NY) and qualified using a Bioanalyzer 2100 (Agilent, Santa Clara, CA). Total RNA is normalized to 7.5ng/ul in a total volume of 50ul on the Perkin Elmer Janus Workstation (Perkin Elmer, Janus II). Poly-A selection and cDNA synthesis are performed using the TruSeq Stranded mRNA kit as outlined by the manufacturer (Illumina, cat#RS-122-2103). All steps are automated on the Perkin Elmer Sciclone NGSx Workstation to reduce batch to batch variability and to increase sample throughput. Final RNASeq libraries are quantified using the Quant-it dsDNA High Sensitivity assay, and library insert size distribution is checked using a fragment analyzer (Advanced Analytical; kit ID DNF474). Samples where adapter dimers constitute more than 4% of the electropherogram area are failed prior to sequencing. Technical controls (K562,Thermo Fisher Scientific, cat# AM7832), are compared to expected results to ensure that batch to batch variability is minimized. Successful libraries are normalized to 10nM for submission to sequencing.