| GSM8410020 channel 1 extraction |
Mouse brain cortex, hippocampus tissue samples, as well as CAD and N2a cell pellets were homogenized in 1mL TRIzol (Invitrogen, 15596018) and incubated at room temperature for 5 min. 200µL Chloroform was added, thoroughly mixed, incubated at room temperature for 15 min, and centrifuged at 13,000g for 20 min at 4ºC. Total RNA was precipitated from 600µL supernatant in 60µL NaAc (3M, pH 5.2), 4µL glycogen (5 mg/mL), and 600µL 100% Isopropanol for at least 30 min at -80ºC. 500ng RNA was reverse transcribed (RT) using SuperScript III First Stand Synthesis System (Invitrogen, 18080051) with random hexamers for downstream qPCR analysis. All RT and qPCR experiments were performed in triplicate and analyzed using the relative quantification (ΔΔCt) method with Actin or Gapdh as the normalization control. All tested primers are provided in Additional File 1. To quantify circRNA levels, divergent primers were designed to span the circRNA back splice junction (BSJ) specifically amplifying the circRNAs and not the counterpart linear RNA. A-tailing RNA was quantified by Qubit RNA High Sensitivity Assay (Thermo Fisher Scientific) and quality was determined by Bioanalyzer 2100 Eukaryote Total RNA Pico (Agilent Technologies). rRNA depleted RNA-seq libraries were generated according to previously described protocols [35]. Briefly, the Ribo-Zero rRNA Removal Kit was used to deplete ribosomal RNAs. Libraries were constructed using the NEBNext UltraTM RNA Library Prep Kit according to the manufacturer’s instructions. Library quality and concentrations were assessed by Tapestation High Sensitivity D1000 ScreenTapes (Agilent Technologies, 5067-5584) and qPCR, respectively. Libraries were equimolarly pooled and sequenced on the Hiseq platform with 150 PE/read length and a target of 80M total reads (40M in each direction) per sample (Admera Health LLC). |
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