title: smd12 developing ear siRNAs, plant 7
design: Small RNA-enriched RNA was extracted from 50 to 100 mg developing ear tissue (4-6 cm stage) using the mirVana miRNA Isolation Kit (Life Technologies; AM1560) with Plant RNA Isolation Aid (Life Technologies; AM9690). 500 ng of each sample was ligated to an adenylated 3’ adapter (0.5 μM) with 5% polyethylene glycol 8000 and 10 units/μL T4 RNA Ligase 2 (truncated K227Q; New England Biolabs; M0351S) in a 10-μL reaction volume at 28°C for 1 hour. The entire product was then directly ligated to an RNA/DNA hybrid 5’ adapter (0.4 μM) with 0.4 units/μL T4 RNA Ligase 1 (New England Biolabs; M0204S) in a 12-μL reaction volume at 28°C for 1 hour. Six μL of the adapter-ligated product was directly reverse transcribed with SuperScript II Reverse Transcriptase (Life Technologies; 18064-014) in a 10-μL volume with 1 μM primer (same indexed reverse primer as used for PCR; see below) at 50°C for 1 hour. Six μL of cDNA was directly PCR-amplified using FailSafe PCR 2X premix buffer (Epicentre; FSP995E), 200 nM forward primer, 100 nM reverse primer (in addition to the reverse primer remaining after reverse transcription), and 1 μL of FailSafe PCR Enzyme Mix (Epicentre; FS99100) in a 50-μL total volume. Thermocycler conditions were as follows: an initial denaturation at 95°C for 1 min; 15 cycles of 95°C for 30 s, 50°C for 30 s, and 68°C for 1 min; and a final extension at 68°C for 7 min. DNA was precipitated in 3 volumes of ethanol, one tenth volume 3 M sodium acetate and 1 μL of 20 mg/ml glycogen. Amplicons of 140 to 150 bp in length were selected by gel electrophoresis using a 4% NuSieve GTG agarose gel (Lonza; 50080). DNA was extracted from the gel with a QIAquick gel extraction kit (Qiagen; 28704) without heating at any stage. Adapter and primer sequences are listed in Supplemental Table 1 of Gent et al (2014) Plant Cell.