Small RNA sequences of Emiliania Huxleyi
E. Huxleyi strain 1516 was grown in artificial seawater with metals and vitamins added to achieve f/2 medium concentrations. Cultures were incubated at 18-20°C under cool white fluorescent light (660 μmol • m2• s2) on a 12 hr light/dark cycle. RNA was then isolated at four, six, eight, ten and twelve days post-inoculation using a standard guanidium isothiocyante procedure with a PEG/NaCl precipitation. 23 μg RNA was loaded in two wells of a 15% denaturing PAGE and the 18-30 nt area of small RNAs was excised. After purification 5’ and 3’-end adaptor sequences were ligated and small RNAs were amplified by using RT-PCR. PCR products were size selected using denaturing PAGE. PCR products of ~92 nt were purified from the gel, and sequenced using the Illumina platform.
EHUW_071107