title: GSM4223656: 4days_rep2_ssRNase_noprotein; Mus musculus; OTHER
library_construction_protocol: Homogenized samples were divided into 2 subsamples one for interrogation of protein binding, the other for interrogation of secondary structure. For protein binding samples, each subsample were then was further divided and half was incubated in either 100 U/ml of a single-stranded RNase (ssRNase) (RNaseONE (Promega; Madison, WI, USA)) with 200 μg/ml BSA in 1X RNaseONE buffer for 1 hour at room temperature (RT), or 2.5 U/ml of a double-stranded RNase (dsRNase) (RNaseV1 (Ambion; Austin, TX, USA)) 1X RNA structure buffer for 1 hour at 37°C as previously described (Anderson et al., 2016). Following RNAse treatment, proteins were denatured and digested by treatment with 1% SDS and 0.1 mg/ml Proteinase K (Roche; Basel, Switzerland) for 15 minutes at RT. Finally, RNAse crosslinks were reversed by incubation at 65°C for 2 hours. For structure only samples, we performed the identical treat-ments as described for protein bound samples, except that the cross-linked homogenate was treated with 1% SDS and 0.1 mg/ml Proteinase K (Roche;Basel,Switzerland)and ethanol precipitated before treatment with the structure specific RNAses. Post digestion, RNA was then isolated using Qiagen miRNeasy RNA isolation kit as described in the manual. Strand specific libraries were constructed as previously described (Silverman et al, 2014).
attributes.tag: GEO Accession
attributes.value: GSM4223656