title: OTTR library of miRXplore miRNA, ddR11biotin_16C_4A, Fig. 7C-D
design: 500 pg of miRXplore miRNA was used as input. 3 labeling began by adding 2  L of Buffer 1A first, followed by  1 L of buffer with 1.0 mM ddATP-11-biotin, 1.0 mM ddGTP-11-biotin, 28 mM MnCl2, 28 mM (NH4)2SO4, 10 mM sodium acetate pH 5.5 and  1 L of labeling enzyme January 2024 W403F753 diluted by 5-fold to 10 M with 140 mM Tris-HCl pH 7.5, 1 M KCl, 14 mM DTT, 35% PEG-8000. 3'-labeling was carried out for 20 hours at 16 C  The final volume after 3 labeling wa 14 L. Unincorporated nucleotides were quenched by 1 L of rSAP and 1 L of Buffer 2 (80 mM MgCl2) for 15 min at 37 C. Cations were chelated by addition of 1 L of Buffer 3 (100 mM EGTA) and rSAP was inactivated by heating the reaction for 5 min at 65 C. cDNA synthesis was performed with 1 L of 10 M July 2023 WT, 1 L of Buffer 4A and 1 L of an oligonucleotide adapter mix containing 1.8 M +T.v2.IR annealed to 1.8 M RNA.v2.1 and 3.6 M c3t_ddCR5N. cDNA synthesis was carried out  for 30 min at 37 C. cDNA size selection after purification was performed by streptavidin pull-down
library_name: ddR11biotin_16C_4A