GSM3580732: AR58_MC_Ndp_R1; Mus musculus; RNA-Seq
6-8 retinas were homogenized in a Dounce homogenizer in 500 μL polysome buffer [50 mM Tris, pH 7.5, 250 mM sucrose, 100 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 200 U/mL RNasin Plus (Promega), 1 mg/mL heparin, 100 g/mL cycloheximide, 0.5 mM spermidine (Sigma),  complete EDTA-free protease inhibitor cocktail (Roche)].  Homogenates were centrifuged at 10,000xg for 10 min to create a postmitochondrial supernatant.  Five μL mouse monoclonal anti-HA antibody (HA.11, ascites fluid; Covance) were added to the supernatants (400 μL) and rotated for 4 hours at 4 °C. 100 μL protein G magnetic beads (Dynabeads; Invitrogen) were washed twice with polysome buffer and added directly to the antibody-coupled polysomes and rotated overnight at 4 °C.  The following day, samples were placed in a magnet on ice and the beads were washed 4x5 minutes in high salt buffer (50 mM Tris, pH 7.5, 300 mM KCl, 12 mM MgCl 2, 1% Nonidet P-40, 1 mM DTT, 20 μg/mL cycloheximide).  To prepare total RNA, 350 μL of Qiagen RLT buffer were added to the beads or to 50 μl of the input samples. Total RNA was prepared according to manufacturer's instructions using RNeasy Plus Micro kit (Qiagen) and the quantity and quality of the RNA were assessed using Bioanalyzer (Agilent). Libraries were constructed using the NuGEN single cell RNA-seq library preparation kit as per the manufacturer's protocol.