title: GSM1614898: LCL19_Polysome; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was isolated with Trizol reagent from the cytoplasm of the three LCLs (total cytosol content), and from pooled polysome sucrose fractions (>3 ribosomes; 250 µL). Two-step column purification and size separation were performed on each RNA sample. First, long RNA (>200 bases) was separated using SpinSmart RNA Purification column (Denville), and the flowthrough (<200 bases) was placed on a second column for small RNA (microRNA) separation with mirPremier microRNA isolation kit (Sigma-Aldrich, St. Louis, MO). RNA quality was evaluated on a Bioanalyzer. Cytoplasmic (total) and polysome derived long RNA (25 ng each) was then converted to cDNA using the NuGen Ovation RNA-Seq System V2 (NuGen Technologies, San Carlos, CA), which uses both random hexamers and oligo-dT to amplify all RNA sequences, while suppressing cDNA formation from ribosomal RNA by >90%. ERCC RNA (External RNA Control Consortium, Ambion) controls were spiked into RNA prior to NuGen cDNA synthesis. The NuGen Ovation RNA-Seq kit produces non-stranded cDNA (3-5 microgram, measured with Qubit (Life Technologies, Foster City, CA)). The double-stranded cDNA derived from long RNAs was sheared to 150-200 bp fragments with a Covaris focused-ultrasonicator (Covaris, Inc. Woburn, MA) and recovered by centrifuging over an YM-30 spin filter (Amicon EMD Millipore Billerica, MA). Fragments longer than 100 bp were retained and eluted from the membrane. We generated barcoded sequencing libraries from 100 ng of sheared cDNA using the NEBNext Fast DNA Library Prep Set for Ion Torrent sequencing (New England Biolabs, NEB, Ipswich, MA), as described [22,48]. In a separate step, purified small RNA fractions (containing 100 ng RNA) were used for library construction using Ion Total RNA-Seq Kit v2 for small RNAs (Life Technologies, Foster City, CA). Pooled barcoded RNA libraries were sequenced on the Ion Torrent Proton platform (Life Technologies, Foster City, CA).
identifiers.namespace: GEO
identifiers.id: GSM1614898