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, ? 1.1µM (in final volume) RNA 5 TB oligonucleotide (5'ae3

, ? 1.1µM (in final volume) RNA 3 TB oligonucleotide (5'ae3' Phosphate

-. and E. , ? 0.55µM (in final volume) DNA SPL5 TB oligonucleotide (5'ae3' CGC

&. and E. , ? 0.55µM (in final volume) DNA SPL3 TB oligonucleotide (5'ae3

K. ?-75mm, Fisherscientific) pipette and was incubated using the following slow cooling hybridization protocol: 95 ¶ C: 1' 70 ¶ C: 3' 65 ¶ C: 3' 60 ¶ C: 3' 55 ¶ C: 3' 50 ¶ C: 3' 45 ¶ C: 3' 40 ¶ C: 3' 35 ¶ C: 3' 30 ¶ C: 3' 25 ¶ C: 3' After hybridization 12µl of the product was used to prepare a reaction solution for RNA oligonucleotides ligation. The protocol of ligation mixture is as follows: ? 1.5µl T4 RNA Ligase 2 (dsRNA ligase, initial concentration 10000units/ml, catalog # M0239S, NEB) mix containing: 1µg RNA lagging strand (purified after splint ligation) 2µg RNA leading strand, Acros Organics

, The reaction solution was homogenized by a pipet and was incubated using the following slow cooling hybridization protocol: 90 ¶ C: 1' 85 ¶ C: 2' 80 ¶ C: 2' 75 ¶ C: 2' 70 ¶ C: 3' 65 ¶ C: 3' 60 ¶ C: 3' 55 ¶ C: 3' 50 ¶ C: 3' 45 ¶ C: 3' 40 ¶ C: 3' 35 ¶ C: 3' 30 ¶ C: 3' 25 ¶ C: 3' Once hybridization was done

, Ligation of sur_RNAbiot_MB Ph The final dsRNA construct, with biotin modifications at its 3 extremities, was obtained by ligating biotin modified sur_RNAbiot_MB Ph RNA oligonucleotide to 3' extremity of the leading strand. For ligation efficiency the ligation reaction reaction solution should contain RNA oligonucleotide with concentration (in molarity)10x prior to dsRNA concentration. The ligation protocol is as follows. For 15µl of reaction solution the following ingredients were mixed: ? 0

-. and Z. , ? 1.6pmol sur_RNAbiot_MB Ph oligonucleotide (5'ae3' Phosphate GCG

, ? 1.5µl T4 RNA Ligase 2 (dsRNA ligase, initial concentration 10000units/ml, catalog # M0239S, NEB)

, ? 1.5µl T4 Rnl2 Reaction Buffer

, Where available, links point to the publisher's website as provided by the doi system

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