title: OTTR library of miRXplore miRNA, Cy5_20pg_1, Fig. 5A-C; Fig. 6F; SI Fig. 3B
design: 20 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 3.5 mM ddATP, 28 mM MnCl2, 28 mM (NH4)2SO4, 10 mM sodium acetate pH 5.5 and  1 L of labeling enzyme Oct 2022 F753 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 90 min at 30 C, followed by an additional 30 min at 30 C after  1 L of 3.5 mM ddGTP was added  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 Oct 2022 WT, 1 L of Buffer 4A and 1 L of an oligonucleotide adapter mix containing 1.44 M +T.v2.Cy5 and 0.36 M +C.v2.Cy5 annealed to 1.8 M RNA.v2 and 3.6 M c3t_CY5N_6RNA. cDNA synthesis was carried out  for 30 min at 37 C. cDNA size selection after purification was for a wide range (10 to 200 nt insert)
library_name: Cy5_20pg_1