title: GSM2302993: hair_rep2_dsRNase_noprotein; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: ~Two million INTACT purified nuclei were lysed in 850 ml RIP buffer (25 mM Tris-HCl, pH = 7.4; 150 mM KCl, 5 mM EDTA, pH = 7.5; 0.5% NP40; 10 mM DTT; 1 tablet protease inhbitors and 0.5ml/ml RNaseOUT (Life Technologies; Carlsbad, CA)) by manual grinding. The resulting cell lysate was treated with RNase-free DNase (Qiagen; Valencia, CA). The lysates were then split and treated with either 100 U/ml of a single-stranded RNase (ssRNase) (RNaseONE (Promega; Madison, WI)) 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)) in 1X RNA structure buffer for 1 hour at 37°C as previously described (Silverman, 2014). See Figure 1A for a schematic representation of library preparation. Proteins were then denatured and digested by treatment with 1% SDS and 0.1 mg/ml Proteinase K (Roche; Basel, Switzerland) for 15 minutes at RT. Proteinase digestion was followed by a 2 hour incubation at 65°C to reverse the RNA-protein cross-links. To determine whether nuclease resistant regions in RNAs are due to protein binding or specific secondary structures, we also determined the digestion patterns of ds- and ssRNases immediately following protein digestion. To do this, we performed the identical treatments as described above except that the cross-linked nuclear lysates were treated with 1% SDS and 0.1 mg/ml Proteinase K (Roche; Basel, Switzerland) and ethanol precipitated prior to being treated with the two RNases. In this way, the SDS and Proteinase K solubilized and digested the proteins allowing us to deduce PPSs within all detectable RNAs in the cells of interest (see Figure 1A for schematic).The digested RNA was then isolated using the Qiagen miRNeasy RNA isolation kit following the included protocol (Qiagen; Valencia, CA). Purified RNA was used as the substrate for strand-specific sequencing library preparation (TruSeq small RNA sequencing kit [illumina, Valencia, CA]), as previously described (Zheng et al. 2010; Li et al. 2011), with the exception that we also include DSN library normalization per manufacturer instructions (Illumina; San Diego, CA).