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The products were size selected, amplified by PCR and sequenced using the conventional SOLiD protocol.
The SOLiD protocol covers a wider range of length, as the gradient of the distribution curve is flatter.
Diversity of these sites near the 3'-end may be caused by the hybridization-based adapter ligation in the SOLiD protocol.
The SOLiD protocol allows simultaneous 5' and 3' adapter ligation to the ends of small RNAs, a method based on hybridization of N6 at the end of the adapters.
The principle of reverse transcription (RT -PCR with stem-loop pRT -PCRs based on hybridization as for the SOLiD protocol, withh could explain the high correlation between qRT-PCR and SOLiD cloning data.
For SOLiD cloning data, the fold difference of miR-1 and miR-206 was similar with Illumina GA data and qRT-PCR, indicating relatively more accurate quantitative measurement for miRNA abundance of the SOLiD protocol.
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The two libraries, bis-sol and bis-gel, were amplified on magnetic beads by emulsion PCR (ePCR) according to standard SOLiD protocols, with the exception that additional dATP and dTTP was added to the aqueous ePCR phase to compensate for the low complexity of the bisulfite converted libraries.
A standard amount of 60 pg for each library was used for separate emulsion PCR reactions (ePCR) following existing SOLiD protocols.
A 10 μg sample of chromosomal DNA was sonicated with the Covaris S2 in order to generate fragments of 80 110 bp to be used for building fragment DNA libraries per existing SOLiD protocols (Foster City, CA).
To further promote DCD transplantation, it is important to share expertise and obtain consensus, so that this can be translated into more uniform and solid protocols supported by the competent authorities, transplant and intensive care professionals, which may eventually result in a further promotion of DCD transplantation in Europe.
Following these conclusions, we feel it is important that experiences with DCD are shared and consensus is obtained, so these can be translated into more uniform and solid protocols supported by the competent authorities, transplant and intensive care professionals, which may eventually result in a further promotion of DCD transplantation in Europe.
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