title: GSM4176150: WT.unlab; Mus musculus; RNA-Seq
library_construction_protocol: Neuronal RNA was extracted by lysing using Trizol directly Five-EU labeled RNA was enriched and RNA-seq library was prepared by adapting the Coller lab (Case Western Reserve University) protocol (personal communication). Specifically, mouse neuron RNA was spiked-in with 10% (w/w) 5-EU labeled fly RNA and 10% (w/w) yeast RNA. The mixed RNA was depleted of rRNA using the Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat) and fragmented using NEBNext® Magnesium RNA Fragmentation Module (NEB, cat. no. E6150S) for 5 min. RNA samples for libraries unlab, A and B were biotinylated by Click-iT chemistry and pulled-down using the Click-iT™ Nascent RNA Capture Kit. The pulled-down samples, together with the input sample, were subjected to library construction the same as for ribosome profiling libraries(Heyer et al., 2015). Here, for the pulled-down samples, all reactions were performed directly on beads until after the reverse transcription step. Sequencing library fraction with insert size 50-200nt was collected and sequenced with Illumina NextSeq RNA labeled with 5EU clicked to biotin, enriched, followed by RNA-seq
identifiers.namespace: GEO
identifiers.id: GSM4176150