Costar spin column

cDNA was mixed with 20 μl gel loading buffer II (Invitrogen), denatured (3 min, 95°C) and placed on ice (3 min). Denatured cDNA was run on 10% TBE-Urea gel (Biorad) (200 V, 50 min) in 1xTBE buffer. The gel was stained with SybrGold (Invitrogen) (5 min, RT) and cDNA corresponding to 80-130 nt was excised. For physical disruption, the gel slices were spun through 0.5 mL tubes with holes at the bottoms nested in 1.5 mL tubes. The disrupted gel slurry was incubated in 400 μl water (10 min, 70°C, 1400 rpm shaking). cDNA was cleared from the gel by transferring the mix into a Costar-spin column (Corning) and centrifuging (20,000 g, 3 min, RT). 25 μl 3 M NaCl, 2 μl Glycoblue and 0.75 mL of isopropanol was added to the cDNA mix. Samples were incubated at −20°C (> 30 min). cDNA was collected by centrifuge (20,000 g, 30 min, 4°C), washed with 0.75 mL cold 80% EtOH and resuspended in 15 μl of 10 mM Tris-HCl (pH 8.0).

Adaptor ligated, and fragmented RNA was mixed with 10 μl gel loading bufferII (Invitrogen), denatured (2 min, 80°C) and placed on ice (3 min). Denatured RNA was run on 10% TBE-Urea gel (Biorad) (200 V, 35 min) in 1 x TBE buffer (diluted from RNase-free 10 X TBE (Ambion)). The gel was stained with SybrGold (Invitrogen) (5 min, RT) and RNA corresponding to 40-90nt was excised. For physical disruption, the gel slices were spun through 0.5 mL tubes with holes at the bottoms nested in 1.5 mL tubes. The disrupted gel slurry was incubated in 600 μl water (10 min, 70°C, 1400rpm shaking). RNA was cleared from the gel by transferring the mix into a Costar-spin column (Corning) and centrifuging (20,000 g, 3 min, RT). 50 μl 3 M Sodium Acetate (pH 5.5), 2 μl Glycoblue and 0.75 mL of isopropanol was added to RNA mix and incubated at −20°C (> 30 min). RNA was collected by centrifugation (20,000 g, 30 min, 4°C), washed with 0.75 mL cold 70% EtOH and resuspended in 10 μl of 10 mM Tris pH 7.0.