GSM5229608: A549_pcdh; Homo sapiens; OTHER
After treated with cycloheximide (CHX, 100 μg/mL) for 15min, cells were washed with CHX/PBS and lysed in polysome lysis buffer (100 mmol/L KCl, 5 mmol/L MgCl2, 10 mmol/L HEPES, 1 mmol/L DTT, 100μg/mL CHX, supplemented with protease inhibitor, RNase inhibitor, and DNase I, pH=7.4), followed by shearing 20 times with 26-gauge needles gently. Lysates were cleared by centrifugation. 10% of cleared lysate was retained as input for total RNA extraction. The remained lysates from the polysome profiling procedure were treated with RNase I (Thermo Fisher Scientific, Waltham, USA) at room temperature for 5 min to obtain ribosome-protected mRNA fragments (RPF). The reaction was stopped by adding SUPERaseIn RNase Inhibitor (Invitrogen, Carlsbad, USA). Subsequently, lysates were loaded onto the 12mL 10-50% sucrose gradients, and ultracentrifuged for 3 h at 35,000rpm, 4˚C on an SW41Ti rotor (Beckman Coulter). 0.75mL fractions were collected using a Brandel Density Gradient Fractionation System with a 254 nm filter lens. Fractions containing 80S ribosome particles were combined and underwent RNA clean-up by TriZol reagent. The RNA sequencing library was prepared according to Ingolia et al.with some modifications. In brief, rRNAs were depleted in both total RNA and RPF RNA samples with the Ribo-Zero Gold kit (Illumina, San Diego, USA). Total RNA was fragmented as input control. Subsequently, the ends of both the RPF RNA and fragmented total RNA samples were repaired prior to 3' and 5' adapter ligation, as described in Illumina Small RNA Library Prep Reference Guide. RNA samples were reverse transcribed by Superscript III reverse transcriptase (Invitrogen, Carlsbad, USA). cDNA libraries then were gel purified and amplified by limited-cycle PCR with index primers. Libraries were cleaned up and subjected to next-generation sequencing on Illumina Hiseq X10 under the PE150 sequencing model.