Blumeria Graminis f. sp. hordei DH14 epiphytic EST sequencing
454FLX sequencing of cDNA extracted from Blumeria graminis f. sp.                 hordei epiphytic mycelia dissected from barley leaves
We dissected epiphytic mycelia from barley leaves                     infected with Blumeria graminis f.sp. hordei using procedures previously                     described [Both, M., et al.Mol Plant-Microbe Interact, 2005. 18: 125-133]. We                     extracted total RNA from the fungal structures obtained from approximately 200                     infected primary leaves using a standard GTC/Phenol protocol [Chomczynsky, P.                     and N. Sacchi. Anal Biochem, 1987. 162: 156-159] followed by clean up with                     RNAClean magnetic affinity beads (Agencourt Bioscience Corp). We obtained about                     150µg total RNA. We then depleted the non-mRNA from the sample using the                     “mRNA-ONLY™ Eukaryotic mRNA Isolation Kit” as follows: we digested 40µg of total                     RNA with “Terminator Exonuclease” in a 120µl reaction at 30°C for 60 minutes and                     stopped the reaction by adding 6µl 100mMEDTA. We precipitated the remaining RNA                     by adding 126 µl 5M LiCl, incubating at – 20°C for 30 minutes and centrifuging                     at 4°C at 13000g in a table-top centrifuge. The pellet was washed twice with 70%                     ethanol, air dried an resupsended in 7.5 µl RNAse-free water. We synthesised                     cDNA following the “ExactSTART™ Full-Length cDNA” protocol described here. The                     5’ CAP was removed from the RNA using TAP in a 10 ul reaction (37 oC for                     30minutes). In the following step we substituted the “acceptor oligo” with a                     custom made oligo-ribonucleotide (Invitrogen) whose sequence was compatible with                     the 454 pyrosequencing procedure (5’GCCUCCCUCGCGUUAUCAGA3’; 50µM final conc) and                     ligated this to the decapped mRNA (final volume 20 µl, incubated at 37°C for 30                     minutes). The cDNA itself was made by using all of this 20 µl ligated RNA                     directly in the 1st strand synthesis reaction as described by the manufacturers,                     except that a custom-made primer (SAD-R poly T: 5’GCCTTGCCAGCCCGCTCCAGT253’;                     50nM final conc) was employed. Likewise the 2nd strand synthesis and                     amplification was carried out using Phusion DNA polymerase (NEB) in a 100 µl                     reaction (95°C 30S; then 18 cycles of 95°C 30S, 60°C 30S, 72°C 4min; ending with                     a 74°C 4 min extension step). The primers used in the amplification reaction                     were modified to render them compatible with the 454/pyrosequencing protocol                     (SAD-For: GCCTCCCTCGCGCCATCAGA, 500nM final conc; SAD-Rev: GCCTTGCCAGCCCGCTCAGT,                     500nM final conc). The overall yield of the resulting cDNA was ca. 7 µ g and gel                     electrophoresis showed a DNA smear with an modal distribution of around 900bp.                     The DNA was sent to ROCHE Diagnostics for 454/pyrosequencing (one run on                     GS-FLX). These yielded 247,306 reads, a total number 50.8M bases, which                     corresponded to an average read length of 205.3 bases.