GSM7431765: PRO12-HUES8-WT; Homo sapiens; RNA-Seq
Around 5 million  hESCs were swelled in swelling buffer (10 mM Tris pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 1XProteinase Inhibitor Cocktail, 10 U/ml Superase-In) for 10mins, then nuclei were extracted in swelling buffer added with 10% glycerol and 0.5% NP-40. The nuclei were resuspended in freezing buffer (50 mM Tris pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 1xPIC, 10U/100uL Superase-IN). Total RNAs from the nuclei were immediately extracted by TRIzol LS, and the NRO RNAs from this mixture were purified by streptavidin C1 beads twice. The purified NRO RNAs were processed to sequencing library using a two rounds of adaptor ligation following eCLIP-seq method with 10nt UMI PRO-seq