Name: GSM7043880
Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Frozen tissue samples were homogenized with pre-chilled Dounce homogenizer (40 strokes using type A pestle, 40 strokes using type B pestle) in cold homogenization buffer on ice [RNAse-free water, 50 mM Tris (Sigma), pH 7.5, 100 mM KCl (Sigma), 12 mM MgCl2 (Sigma), 1% Nonidet P-40 (Sigma), 200 U/mL RNAsin (Promega, catalog #N2115), 1 mM DTT (Sigma-Aldrich), proteinase inhibitors (Roche), 1 mg/mL heparin (Sigma-Aldrich), 0.1 mg/mL cyclohexamide (Sigma-Aldrich)] to a wt/vol ratio of ~2-5%. Total RNA samples were also pooled respective to the OL samples for consistency. Homogenates were centrifuged at 10,000 x g for 10 minutes at 4°C to remove excess tissue. 10% of the cleared lysate was taken as input (IN, total spinal cord RNA) and stored at -80°C for subsequent analysis. The remaining lysate was incubated with monoclonal mouse anti-hemagglutinin (HA) antibody (HA.11 Clone 1612) for 4-6 h at 4°C in a microtube rotator and end-over-end gentle mixing. Protein A/G magnetic beads (Pierce) were prewashed in homogenization buffer and added to the lysates and further incubated at 4°C overnight with end-over-end mixing. The following day, samples were placed in a magnetic microtube stand on ice to isolate the magnetic beads, and the supernatant was stored in -80°C. Magnetic beads with the bound immunoprecipitated mRNA (IP) were washed 3 times using ice-cold high salt buffer solution (50 mM Tris, pH 7.5, 300 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 100 g/mL cycloheximide) for at least 5 mins at 4°C and end-to-end mixing. Input and immunoprecipitated mRNA were purified using RNeasy Mini, RNA isolation kit (Qiagen, catalog # 74104) and Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, catalog # LSKIT0204) according to the manufacturers' protocol, respectively. Concentration and quality of isolated RNA was first determined using a NanoDrop 1,000 spectrophotometer (Thermo Scientific). Then, the concentration was measured by Qubit Fluorometer (Thermo Scientific), and RNA integrity was assessed by capillary electrophoresis (Bioanalyzer, Agilent Technologies). Libraries were prepared using the Universal Plus mRNA-Seq (NuGEN Cat# 0508).