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Fragments were purified using AMPureXP beads (Beckman Coulter Inc, Brae, CA, USA) to remove small products (<100 bp), yielding 1 <span class="lh">ug of material which was end-polished, A-tailed and adapter-ligated according to the manufacturer's protocol.
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One-tenth (2.5 ug) of starting material was kept aside as input DNA control.
Briefly, following first strand and second strand cDNA synthesis, samples underwent a single round (10 ug starting material) or two rounds (10 50 ng starting material) of linear amplification using a T7 based in vitro transcription (IVT) kit (MegascriptT7, Ambion).
Therefore, the in solution method is preferred for larger DNA input (>2 ug starting material), while the in gel method is ideal for low amounts of starting material.
0.5 ug of total RNA was used as starting material for 1 round of aRNA amplification, resulting in 26 ug and 51 ug of aRNA from leaves and buds, respectively.
The amount of single stranded material recovered from the 100 ng starting material was 10x less than that recovered from the 1 ug of starting DNA.
We suspected that it could be either the effects of BNP was saturated at 2 ug or a reversing effect developed at 3 ug of BNP.
For immunoprecipitation (IP), 500 ug of total lysate was precleared for 1 hour.
Introduction of air with a superficial velocity, Ug, of 1.92 mm/s yielded a layer inversion velocity at UL=30.4 mm/s.
Approximately 2 ug of total RNAs were used for reverse transcription using the Reverse Transcription System (Promega, USA).
20 ug of amplification products were precipitated and mixed with 100 ug COT-1 DNA (Roche).
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