PTRE-seq in N2A
Next generation sequencing library preparation was performed as previously described (Kwasnieski et al. 2012; White et al. 2013; Kwasnieski et al. 2014). RNA was isolated from cells using RNeasy Kit (Qiagen) or from polysome fractions with Trizol (Amresco).  RNA was treated with Turbo DNase (Life Technologies) and reverse transcription was performed using Superscript IV reverse transcriptase (Life Technologies). The 3  UTR of the luciferase gene containing barcode sequences was amplified (Phusion high fidelity master mix, New England Biolabs) from cellular or polysome associated RNA (cDNA) or plasmid DNA prior to next-generation sequencing. Each experiment was sequenced as a pool on four lanes on an Illumina MiSeq sequencer as a spike-in. Illumina Single End