title: GSM8195855: SCR, scramble gRNA, rep 3; Homo sapiens; OTHER
library_construction_protocol: RNA was extracted using TRIzol LS reagent (Invitrogen). RNAs were diluted into formamide load buffer, heated to 95°C for 3 minutes, and fractionated on a 10% polyacrylamide, Tris-Borate-EDTA (TBE) gel containing 7M urea. Sybr Gold nucleic acid staining (Invitrogen) was conducted to identify the RNA pattern. For primer extension analysis, 1.5 μg of total RNA was pre-annealed with 5'-32P-labeled oligonucleotide and 5x hybridization buffer (250 mM Tris, pH 8.5, and 300 mM NaCl) in a total volume of 7 μl. The mixture was heated at 95°C for 3 min followed by slow cooling to 42°C. An equal amount of extension mix consisting of avian myeloblastosis virus reverse transcriptase (Promega), 5x AMV buffer and 40 μM dNTPs was added. The mixture was then incubated at 42°C for 1 hour and loaded on 15% 7M urea denaturing polyacrylamide gel. Gels were exposed on a phosphor screen (GE Healthcare) and scanned on a Bio-Rad personal molecular imager followed by analysis using NIH ImageJ software. Primer extension oligonucleotide sequences were previously described (Dewe et al., 2017). RNA 3' de-phosphorylation was carried out as previously described (Huppertz et al., 2014) using 500 ng of total RNA from each sample. Briefly, samples were treated with T4 Polynucleotide Kinase (T4PNK; New England Biolabs) in a modified 5x reaction buffer (350 mM Tris-HCl, pH 6.5, 50 mM MgCl2, 5mM dithiothreitol) under low pH conditions in the absence of ATP for 30 mins. AlkB treatment was performed as previously described (Cozen et al. Nat Methods. 2015). OTTR-seq libraries were generated as previously described (Upton et al. 2021). Briefly, total PNK-treated RNA was 3' tailed using mutant BoMoC RT in buffer containing only ddATP for 90 minutes at 30 ̊C, with the addition of ddGTP for another 30 minutes at 30 ̊C. This was then heat-inactivated at 65 ̊C for 5 minutes, and unincorporated ddATP/ddGTP was hydrolyzed by incubation in 5 mM MgCl2 and 0.5 units of shrimp alkaline phosphatase (rSAP) at 37 ̊C for 15 minutes. 5 mM EGTA was added and incubated at 65 ̊C for 5 minutes to stop this reaction. Reverse transcription was then performed at 37 ̊C for 30 minutes, followed by heat inactivation at 70 ̊C for 5 minutes. The remaining RNA and RNA/DNA hybrids were then degraded using 1 unit of RNase A at 50 ̊C for 10 minutes. cDNA was then cleaned up using a MinElute Reaction CleanUp Kit (Qiagen). To reduce adaptor dimers, cDNA was run on a 9% UREA page gel, and the size range of interest was cut out and eluted into gel extraction buffer (300mM NaCl, 10mM Tris; pH 8.0, 1mM EDTA, 0.25% SDS) and concentrated using EtOH precipitation. Size-selected cDNA was then PCR amplified for 12 cycles using Q5 High-fidelity polymerase (NEB #M0491S). Amplified libraries were then run on a 6% TBE gel, and the size range of interest was extracted to reduce adaptor dimers further. Gel slices were eluted into gel extraction buffer (300mM NaCl, 10mM Tris; pH 8.0, 1mM EDTA) followed by concentration using EtOH precipitation. Final libraries were pooled and sequenced on an Illumina NextSeq 500 150-cycle high-output kit.