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LincGET RNA was modified both in embryos and in vitro. Briefly, for in-cell SHAPE probing, LincGET were first in vitro transcribed and biotinylated with Pierce RNA 3'-End Desthiobiotinylation Kit (Pierce, #20163). Biotin-labeled LincGET and pre-mRNAs for NONO/PSPC1/CARM1/hnRNPU were injected into 1-cell embryos at phCG 25h, and moved into a 50 µL PBS micro-drop containing 200 mM In Vivo SHAPE Reagent 2-methylnicotinic acid imidazolide (NAI) (Sigma, 03-310), after washing three times with PBS at phCG 48h. Embryos were then incubated at 37°C for 15 minutes. After lysis, biotin-labeled LincGET was purified through MyOne Streptavidin C1 beads (Invitrogen, 65001). For in vitro probing, in vitro transcribed LincGET was re-folded in folding buffer (100 mM NaCl, 100 mM HEPES pH 8.0, and 10 mM MgCl2 in water) at 37°C for 20 minutes. As a control, one group of LincGET was denatured in Denaturing Control Buffer (50% formamide, 50 mM HEPES pH 8.0, and 4 mM EDTA pH 8.0 in water) at 95°C for 1 minutes. For each group, approximately 5 µg RNA was then added to one-ninth volume of NMIA (Invitrogen, M25) at 100 mM in neat DMSO (10 mM final concentration), and incubated at 37°C for 22 minutes. For both in-cell and in vitro probing, background was assessed by performing no-reagent and denaturing controls. After fragmentation with RNA fragmentation reagent (Ambion, AM8740), modified LincGET was subjected to mutational profiling (MaP) reverse transcription (15), with SuperScript II Reverse Transcriptase (Invitrogen, 18064014) under Mn2+ condition (50 mM Tris HCl pH 8.0, 75 mM KCl, 10 mM DTT, 2 mM dNTPs, and 15 mM MnCl2 in water) using random nonamer primers (200 ng/μL, NEB, S1254S). After synthesizing the second strand by NEBNext pre-mRNAs Second Strand Synthesis Module (NEB, E6115S), the resulting cDNAs were constructed for high-throughput sequencing libraries and sequenced by BGI company. |
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RT-PCR |
PAIRED
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