tissue: ~250 micrometers root tip stage: 13-14 day old seedlings 3' adapter: TTTAACCGCGAATTCCAG
Extracted molecule
total RNA
Extraction protocol
Small RNA libraries were constructed using a method that requires the 5’ end of the small RNAs to be phosphorylated [Lau, N.C., Lim, L.P., Weinstein, E.G., Bartel, D.P. (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans Science, 294, 858–862]. Total cellular RNA was fractionated on a 15% acrylamide gel run in 8M urea and 1X TBE, and the region of the gel containing 16 to 28 nt RNAs was excised. The small RNAs were eluted in 0.3 M NaCl and ethanol precipitated with 2.8 volumes of ethanol. The small RNAs were ligated to one of three alternative RNA-DNA chimeric pre-adenylylated 3´-adaptor oligonucleotides (see elsewhere for specific adapter) in a reaction containing 20 uM 3´-adaptor, 50 mM Hepes pH 8.3, 5 mM MgCl2, 3 mM DTT, 5 ug/ ml BSA and 8% glycerol for 2 hr. The ligated small RNAs were gel isolated as described above and ligated to an RNA 5’ adaptor (ATCGTAGGCACCTGAAA) and subsequently gel isolated. The resulting RNAs were reversed transcribed and amplified by PCR. The resulting DNAs were gel purified and sequenced by 454 Life Sciences.
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
size fractionation
Instrument model
454 GS
Description
18-34nt small RNAs
Data processing
5' and 3' adapter sequences were identified by alignment, and RNA sequences between these were extracted. Often more than one RNA sequence could be obtained from a single 100nt sequence read.
