GSM4745450: MEF cells-RiboSeq_HF2; Mus musculus; OTHER
For Total RNASeq, RNA was extracted with Trizol reagent according to manufacturer's protocol. For RiboSeq, lysate was digested with 100 units of RNase I (Ambion) at 4°C for 1 h with mild agitation. Sample digestion was quenched by adding 200 units of SUPERase·IN (Ambion). Digested samples were then placed on top of 10 –50% sucrose gradients supplemented with SUPERase·IN and subjected to ultracentrifugation in a Beckman SW41Ti rotor at 40,000 rpm for 2 h at 4°C. Following centrifugation, the fractions corresponding to the monosome peak of the digested samples were collected using a Biocomp piston gradient fractionator coupled with a Gilson fraction collector and stored in TRIzol LS reagent. RFPs and total RNA were collected using TRIzol reagent. Total RNA was fragmented by alkaline hydrolysis. RFPs and fragmented RNA were collected by gel extraction from a 15% denaturing TBE-urea gel (Invitrogen). For total RNA sequencing: total RNA extracted by trizol was fragmented by alkaline hydrolysis and run on 15% denaturing TBE gel then extracted and barcoded sequencing adapters were ligated. For RiboSeq: Protocol from Ingolia Nat Methods .2017 (doi:10.1016/j.ymeth.2017.05.028) was followed with ribosomal RNA depletion performed with Ambion Ribominus kit.