Similar protocols

Protocol publication

[…] pbio.dundee.ac.uk/kinomer. When contradictions occurred, a final determination was made by phylogenetic analysis against representative kinases., Proteins were searched for matches against the Pfam and SMART databases http://pfam.sanger.ac.uk, http://smart.embl-heidelberg.de using an E threshold of 10-5. P. infestans gene models having weak matches to the protein kinase domain (E > 10-10) were rechecked to help eliminate errors. Transmembrane domains were identified using the TMHMM server at http://www.cbs.dtu.dk/services/TMHMM/, and signal peptides using PSORT at http://psort.ims.u-tokyo.ac.jp/form.html., Alignments of protein sequences were performed using the SEAVIEW implementation of MUSCLE using default parameters []. Maximum likelihood trees were made from these alignments using PhyML, using the LG substitution model and collecting SH-like aLRT data for branch support. These were compared with neighbor-joining trees constructed using BioNJ using 100 bootstrap replicates for alignments of the total kinome, or 500 replicates for individual groups of kinases. Trees were visualized using the FigTree program., mRNA levels during development were calculated from Affymetrix microarray data [], which are deposited in NCBI GEO as series GSE9623, or data generated for this study by qRT-PCR. This employed DNAse-treated RNA from nonsporulating vegetative hyphae grown on rye-sucrose broth, freshly harvested and unchilled sporangia from 7-day cultures, and swimming zoospores released from the sporangia prepared as described []. At least two biological replicates of each tissue were used. Hot-start Taq polymerase (Applied Biosystems, Foster City, California USA) was used in amplifications with primers targeted to the 3' portions of genes (150-225 nt amplicons; Additional File Table S4), w […]

Pipeline specifications

Software tools MUSCLE, PhyML, FigTree
Databases Kinomer