GSM4237563: Control IgG 4; Mus musculus; RNA-Seq
Fresh retinas were dissected, snap-frozen into liquid nitrogen, homogenized in polysome buffer (50 mM Tris pH 7.5, 100 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 200 U/ml RNase inhibitor, protease inhibitor cocktail (P2714, Sigma), 1 mg/ml heparin, and 100 mg/ml cycloheximide) with pellet pestles, and centrifuged at 4°C for 10 min with 12,000 g. Ten% of supernatant was reserved as an 'input' and immediately stored at -80°C. The remaining supernatant was incubated with magnetic beads with anti-HA antibody (M180-9, MBL) at 4°C. After the supernatant was aspirated, the beads were washed with high salt buffer (50 mM Tris pH 7.5, 300 mM KCl, 12 mM MgCl2, 1 mM DTT, and 100 mg/ml cycloheximide) and re-suspended with supplemented RLT buffer (400 µl, with β-mercaptoethanol, Qiagen). The suspensions were used for 'HA-enriched' RNA extraction. RNAs from both 'input' and 'HA enrichment' were extracted using RNeasy Plus Mini kit (Qiagen) according to the manufacturer's instruction. Total RNA quality was assessed with the Agilent 2100 Bioanalyzer System. Extracted RNA samples were processed using the QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen, Vienna) and sequenced on an Illumina NextSeq 500. A median 2.0x107 (range 1.7x107 to 2.4x107) single-end reads with 75 bp were generated.