title: GSM4291977: ppp-1(wrm15) polysomal RNA replicate 3; Caenorhabditis elegans; RNA-Seq
library_construction_protocol: Synchronized gravid day 1 adults were grown on NGM plated seeded with OP50. Per genotype and replicate, ~12000 worms were harvested and washed twice with M9, once with M9 supplemented with 1 mM cycloheximide (Sigma) and once with lysis buffer (20 mM Tris pH 8.5, 140 mM KCl, 1.5 mM MgCl2, 0.5 % Nonidet P40, 1 mM DTT, 1 mM cycloheximide). Worms were pelleted and resuspended in 350 µL cold lysis buffer supplemented with 1% sodiumdeoxycholate (DOC, Sigma). Resuspended worms were lysed using a chilled Dounce homogenizer. Ribonuclease inhibitor RNasin (Promega) was added to samples at a concentration of 0,4 U/µL. Samples were then mixed and incubated on ice for 30 min, followed by a centrifugation step (12000 g, 10 minutes, 4°C) for clearance. The pellet was discarded and the RNA concentration of the supernatant was estimated by absorbance measurement at 260 nm.  To prepare sucrose gradients, 15% (w/v) and 60% (w/v) sucrose solutions were prepared in basic lysis buffer (20mM Tris pH 8.5, 140 mM KCl, 1.5 mM MgCl2, 1mM DTT, 1mM cycloheximide). Linear sucrose gradients were produced using a Gradient Master (Biocomp). Equivalent amounts of sample (around 400 µg RNA) were loaded on the gradient and centrifuged at 39000 g for 3 h at 4°C, using an Optima L-100 XP Ultracentrifuge (Beckman Coulter) and the SW41Ti rotor. To analyze the sample on the gradient during fractionation, absorbance at 254 nm was measured and recorded (Econo UV monitor EM-1, Biorad) using the Gradient Profiler software (version 2.07). Gradient fractionation was performed from the top down using a Piston Gradient Fractionator (Biocomp) and a fraction collector (Model 2110, Biorad). For precise analysis of ribosomal fractions, they were collected by hand according to their absorbance profile; for RNAseq and qPCR analyses, one fraction for 80S ribosomes and one for polysomes (excluding disomes) was collected per sample. RNA extraction from total lysates and from each fraction was performed using the Direct-zol RNA MicroPrep Kit (Zymo Research) according to the manufacturer´s recommendations. RNA libraries were prepared for sequencing using standard Illumina protocols with a ribosomal RNA depletion method by Cologne Center for Genomics.
attributes.tag: GEO Accession
attributes.value: GSM4291977