title: GSM7783500: Excell_SHAPE_rep2_amp2; Homo sapiens; OTHER
library_construction_protocol: For in cellulo probing, LNCaP cells were treated with 25 mM 5-nitroisatoic anhydride (5NIA) in 6-well plates. RNA was isolated by TRIzol followed by treatment with DNAse I. For ex cellulo probing, RNA was isolated following previous protocols (10.1021/acs.biochem.5b00977) before 5NIA treatment. For in vitro probing, 5NIA treatment occured after isolation of RNA. For in vitro and ex cellulo probing, RNA was incubated in 50 mM K-HEPES pH 7.5, 150 mM KCl, 5 mM MgCl2, and 0.1 mM EDTA final. RNA was reverse transcribed and amplified using targeted amplicons tiling the SChLAP1 transcript. Library prep was performed using a 2-step PCR approach (10.1038/nprot.2015.103) DNA-seq of RT-PCR-derived targeted sequencing libraries for SChLAP1. For in cellulo and ex cellulo SChLAP1, amplicons were independently barcoded. For in vitro SChLAP1, amplicons from a given condition (e.g. DMSO or SHAPE) were barcoded identically and pooled together for sequencing.