title: GSM4497767: pycr-1(wrm22) replicate 2 Poly RNA; 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 fractions containing 3 ribosomes per mRNA or more were 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: GSM4497767