UTR introns, antisense RNA and differentially spliced transcripts between Plasmodium yoelii subspecies

Additional file 1. Evaluation of RNA quality from the two NSM parasite samples in agarose gel (a), and a flow chart of data processing and analysis (b)

GOFAST: An Integrated Approach for Efficient and Comprehensive Membrane Proteome Analysis

Membrane proteomics, the large-scale analysis of membrane proteins, is often constrained by the difficulties of achieving fully resolvable separation and resistance to proteolysis, both of which could lead to low recovery and low identification rates of membrane proteins. Here, we introduce a novel integrated approach, GELFrEE Optimized FASP Technology (GOFAST) for large-scale and comprehensive membrane proteins analysis. Using an array of sample preparation techniques including gel-eluted liquid fraction entrapment electrophoresis (GELFrEE), filter-aided sample preparation (FASP), and microwave-assisted on-filter enzymatic digestion, we identified 2 090 proteins from the membrane fraction of a leukemia cell line (K562). Of these, 37% are annotated as membrane proteins according to gene ontology analysis, resulting in the largest membrane proteome of leukemia cells reported to date. Our approach combines the advantages of GELFrEE high-loading capacity, gel-free separation, efficient depletion of detergents, and microwave-assisted on-filter digestion, minimizing sample losses and maximizing MS-detectable sequence coverage of individual proteins. In addition, this approach also shows great potential for the identification of alternative splicing products

Functional annotation clustering of the 489 subset from RNA sequencing results.

<p>The Gene Ontology cluster analysis was performed using the GO Fat database (GOTERM_BP_FAT), developed as part of the Annotation Tool of the DAVID suite of bioinformatics resources. The clusters with enrichment scores greater than 1.4 and showing <i>p</i> value<0.05 are presented. The number of genes representing each of the GO terms is given with associated p value and false discovery rate (FDR).</p

Spot assays to determine the effect of various inhibitors.

<p>Cells were serially diluted and spotted onto media containing the indicated inhibitors and incubated at 30°C in 20% CO₂ for 3 days or in 1% O₂ at 30°C for 3 days. (A) Ergosterol biosynthesis inhibitors: 8 µg/ml fluconazole (Flu) and 0.25 µg/ml fenpropimorph (Fen). (B) Histone deacetylase inhibitors: 64 µg/ml trichostatin A (TSA) and 128 mM sodium butyrate (NaB). (C) RNA processing inhibitors: 2 mM amiloride hydrochloride hydrate (Ami) and 5 µg/ml staurosporine (Sta). (D) Protein translation inhibitors: 0.1 µg/ml cycloheximide (Cyc) and 40 mg/ml streptomycin (Str).</p

Hog1 is not hyperphosphorylated in <i>Δptp3Δ</i>, <i>ras1Δ</i>, and <i>cdc24</i>.

<p>(A, B, and C) Cells of the indicated strains were grown to early logarithmic phase, exposed to 1% O₂ for 1 hour in YPD medium and total protein extracts were analyzed by Western blot analysis. The phosphorylation status of Hog1 was monitored using anti-phospho-specific p38 antibody (P-Hog1). The same blots were stripped and reacted with anti-Hog1 antibody for Hog1 loading control (Hog1). Levels of Hog1 phosphorylation were quantitated and expressed as relative levels to the control wild-type H99 grown at 20% O₂. The bar represents standard deviation of four biological repeats. The * indicates p<0.05 compared to H99 at 20% O₂.</p

Spot assay for growth phenotype.

<p>Three-fold serial dilutions of each strain were spotted on YEPD medium and incubated at indicated conditions. Suppressor test in <i>cdc24Δ</i> (A) or <i>ras1Δ</i> (B). Two independent transformants of <i>cdc24Δ</i> and <i>ras1Δ</i> were tested. The cultures were incubated in 20% O₂ at 30°C for 3 days or in 1% O₂ at 30°C for 5 days. (C, D and E) Growth phenotype of various mutants. The cultures were incubated in 20% O₂ at 30°C for 3 days or in 1% O₂ at 30°C for 3 (C and E) or 5 days (D). Growth assays presented in each panel of the composite images of A–D were performed on the same day. (F) Phenotype of <i>ras1Δcdc24Δ</i> double deletant. The cultures were incubated either at 28°C, 37°C, 20% O₂ with 5% CO₂ at 28°C, or 5% O₂ with 5% CO₂ at 28°C for 3 days, respectively. (G) Phenotype of <i>cdc24Δptp3Δ</i> double deletant. The cultures were incubated either at 28°C, 37°C, 20% O₂ with 5% CO₂ at 28°C, or 6% O₂ without CO₂ at 28°C for 3 days, respectively.</p

Functional annotation clustering of the 441 subset from RNA sequencing results.

<p>The Gene Ontology cluster analysis was performed using the GO Fat database (GOTERM_BP_FAT), developed as part of the Annotation Tool of the DAVID suite of bioinformatics resources. Enrichment score of 1.3 is equivalent to non-log scale <i>p</i> = 0.05 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004292#pgen.1004292-Huangda1" target="_blank">[38]</a>. The clusters with enrichment scores greater than 1.4 and <i>p</i> value<0.05 are presented. The number of genes representing each of the GO terms is given with associated <i>p</i> value and false discovery rate (FDR).</p

Sterol profile analysis.

<p>(A and B) Sterols of each indicated strain were extracted from cells grown in 20% or 1% O₂ for indicated time points. Sterol profiles were analyzed by gas chromatography/mass spectrometry. The amount of each sterol is expressed as the relative ratio to cholesterol, the internal recovery standard. Only the major ergosterol intermediates (mean relative amount >0.1 according to <i>cdc24Δ</i> in 20% O₂) are shown. The results of discernable minor intermediates (mean relative amount <0.1 according to <i>cdc24Δ</i> in 20% O₂) are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004292#pgen.1004292.s004" target="_blank">Figure S4</a>. Data were derived from three biological repeats. Statistical <i>t</i>-test was performed by comparing each mutant from each time point to the corresponding wild-type. The * represents <i>p</i><0.05.</p