GSM1251883: HeLa_NO_SSase_plusP_rep1; Homo sapiens; OTHER
We lysed the cell pellets using 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/10mL) and manual grinding. The resulting cell lysate was treated with RNase-free DNase (Qiagen; Valencia, CA). Subsequently, these DNA-depleted lysates were treated with either a single-stranded RNase (ssRNase) (RNaseONE (Promega; Madison, WI)) or double-stranded RNase (dsRNase) (RNaseV1 (Ambion; Austin, TX)) as previously described ((Zheng et al. 2010; Li et al. 2011), see Fig. 1A for a schematic description). Proteins were then denatured and digested by treatment with 1% SDS and 0.1mg/ml Proteinase K (Roche; Basel, Switzerland). It is worth noting for clarity that we had two cell lysates for these experiments: one treated with the ssRNase and the other with dsRNase. For formaldehyde cross-linking experiments, Proteinase digestion was followed by a 2-hour incubation at 65°C to reverse the crosslinks, whereas for UV cross-linking experiments, RNA was liberated from protein by retreating the lysates with 1% SDS and 1 mg/ml Proteinase K for 30 minutes. 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 in the absence of bound proteins. To do this, we performed the identical treatments as described directly above except that the cross-linked cellular 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 protein-protected sites within all detectable RNAs in the cells of interest (see Fig. 1 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).
GEO Accession
GSM1251883