title: GSM2974949: mNET_total_rep1; Arabidopsis thaliana; OTHER
library_construction_protocol: The tissue powder was solubilized in 30 ml lysis buffer (50 mM HEPES, pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 5 mM 2-mercaptoethanol, 2 μg/ml pepstatin A, 2 μg/ml aprotinin, 1 mM PMSF), mixed well and filtrated through 110 μm nylon mesh and a 40 μm cell strainer.  The nuclei were spanned down at 3,200 g for 20 min at 4°C, followed by two washes with HBB buffer (25 mM Tris–HCl, pH 7.6, 0.44 M sucrose, 10 mM MgCl2, 0.1% Triton-X, 10 mM 2-mercaptoethanol) and HBC buffer (20 mM Tris–HCl, pH 7.5, 352 mM sucrose, 8 mM MgCl2, 0.08% Triton-X, 8 mM 2-mercaptoethanol, 20% glycerol). The obtained nuclei pellet were resuspended with 1 ml MNase buffer (20 mM Tris–HCl, pH 8.0, 5 mM NaCl, and 2.5 mM CaCl2) and digested with 20 U MNase (TaKaRa, 20U/μl) for 5 minutes at 37°C rotating at 1,400 rpm. Stopped the digestion by adding 40 μl 500 mM EDTA. Pol II-nascent RNA complex was released by mild sonication. Supernatants were collected by centrifuging at 16,000 g for 5 min at 4°C and Pol II complex were immunoprecipitated according to Nojima et al. [14]. The Pol II bond nascent RNAs were treated by T4 PNK on beads and extracted with 500μl TRIzol Reagent (Invitogen). The IP RNA was resolved on an 8% TBE-urea polyacrylamide gel and RNA with size from 35-100 nt was recovered. The 35-100 nt IP RNA was used to construct the NET-SEQ cDNA library with NEXTflex™ Small RNA-Seq Kit v3 (Bioo Scientific) according to the manual. The cDNA library was resolved in a non-denaturing 6% TBE polyacrylamide gel and size region 150-230 bp was recovered. cDNA library concentration was determined by Qubit QuantIT (Invitrogen) and kapa quantitative realtime PCR.
attributes.tag: GEO Accession
attributes.value: GSM2974949