GSM4869086: Input4_replicate1: SH-SY5Y LSM12 shRNA total RNA; Homo sapiens; RNA-Seq
SH SY5Y cells stably expressing GFP-RPL10A were collected and lysed in a lysis buffer (20 mM HEPES-KOH pH 7.4, 150 mM KCl, 10 mM MgCl2, 1%(v/v) NP-40, 100 μg/ml cycloheximide, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and 200 U/ml RNase inhibitor) at 4C for 15 minutes with gentle rocking. Lysates were first centrifuged at 2,000 x g for 5 minutes at 4C, and the supernatant was collected and further clarified by centrifugation at 20,000 x g for 10 minutes at 4C. After clarification by centrifugation, 30 ul of soluble extracts were retained as input, and the remaining soluble extract was incubated with anti-GFP antibody (NeuroMab) at 4C for 1.5 hours with gentle rocking. Pre-equilibrated Protein G magnetic beads (New England BioLabs) were added to the extracts and further incubated at 4C for 1.5 hours with gentle rocking. The beads were washed four times with a wash buffer (20 mM HEPES-KOH pH 7.4, 350 mM KCl, 5 mM MgCl2, 1%(v/v) NP-40, 100 μg/ml cycloheximide, 1 mM DTT, 1 mM PMSF, and 200 U/ml RNase inhibitor) prior to RNA extraction. Input and translating ribosome-associated RNAs were purified using an RNeasy Mini kit (QIAGEN) according to the manufacturer's instructions. Ribosomal RNA was depleted from 50 ng of purified RNA samples using an rRNA depletion kit (New England BioLabs). cDNA libraries for RNA sequencing were generated using a NEBNext Ultra Directional RNA Library Prep Kit for Illumina according to the manufacturer's instructions (New England BioLabs).