Project description:QuantSeq-Rev method to generate highly strand-specific next-generation sequencing (NGS) libraries enabling transcript quantification and identification of the 3'end of polyadenylated RNAs
Project description:Nascent RNAseq in conjunction with Illumina TRUseq method to sequence total RNAs including short lived RNAs using highly strand-specific next-generation sequencing (NGS) libraries
Project description:QuantSeq-Rev method to generate highly strand-specific next-generation sequencing (NGS) libraries enabling transcript quantification and identification of the 3'end of polyadenylated RNAs
Project description:Proteomic analysis of differentially expressed proteins in MDA-MB-231 and MCF-10A cell lines when miR-200c and miR-203 were transiently expressed or inhibited, respectively.
Project description:In this study, we make used of mRNA-seq and its ability to reliably quantify isoforms, integrating this data with ribosome profiling and LC-MS/MS, to assign ribosome footprints and peptides at the isoform level. We leverage the principle that most cell types, and even tissues, predominantly express a single principal isoform to set isoform-level mRNA-seq quantifications as priors to guide and improve allocation of footprints or peptides to isoforms. Through tightly integrated mRNAseq, ribosome footprinting and/or LC-MS/MS proteomics we demonstrate that a principal isoform can be identified in over 80% of gene products in homogenous HEK293 cell culture and over 70% of proteins detected in complex human brain tissue. Defining isoforms in experiments with matched RNA-seq and translatomic/proteomic data increases the functional relevance of such datasets and will further broaden our understanding of multi-level control of gene expression. In this PRIDE submission you will find the raw files for the HEK293 cell proteomics. Files for the human brain proteomics can be found at PXD005445. We have also uploaded a zip file that contains the input files for our HEK293 cell analysis, and the isoform level output files – there is a separate folder within the zip files for these. The data used to create the manuscript figures is in the Rdata file. Code for assigning peptides and footprints to isoforms can be found on Github here: https://github.com/rkitchen/EMpire
Project description:Pseudomonas aeruginosa is an opportunistic pathogen that requires iron for growth and virulence, yet this nutrient is sequestered by the innate immune system during infection. When iron is limiting, P. aeruginosa expresses the PrrF1 and PrrF2 small regulatory RNAs (sRNAs), which post-transcriptionally repress expression of non-essential iron-containing proteins thus sparing this nutrient for more critical processes.The genes for the PrrF1 and PrrF2 sRNAs are arranged in tandem on the chromosome, allowing for the transcription of a longer heme-responsive sRNA, termed PrrH. While the functions of PrrF1 and PrrF2 have been studied extensively, the role of PrrH in P. aeruginosa physiology and virulence is not well understood. In this study, we performed transcriptomic and proteomic studies to identify the PrrH regulon.
Project description:Organic acid secretion is a widespread physiological response of plants to alkalinity. However, the features of alkali-induced secretion of organic acid and the underlying mechanism are poorly understood. Herein, it was indicated that oxalate was the main organic acid synthesized and secreted in vine roots, and acetate synthesis and malate secretion were also promoted under NaHCO3 stress. NaHCO3 stress enhanced H+ efflux rate of vine roots, which was related to plasma membrane H+-ATPase activity. Transcriptomic profiling revealed that carbohydrate metabolism was the most significantly altered biological process under NaHCO3 stress; a total of seven genes related to organic acid metabolism were significantly altered, including two PEPCs and PEPCKs. Additionally, the expression levels of five ABC transporters, particularly ABCB19, as well as two malate transporter ALMT2s, were largely upregulated by NaHCO3 stress. Phosphoproteomic profiling demonstrated that the altered phosphoproteins were primarily related to binding, catalytic activity and transporter activity in light of their molecular functions. The phosphorylation levels of PEPC3, two plasma membrane H+-ATPases 4 and ABC transporters ABCB19 and PDR12 were significantly increased. Additionally, the inhibition of ethylene synthesis and perception completely blocked NaHCO3-induced organic acid secretion, while the inhibition of IAA synthesis reduced NaHCO3-induced organic acid secretion. Collectively, our results demonstrated oxalate as the main organic acid under alkali stress and the necessity of ethylene in mediating organic acid secretion, as well as further identified several candidate genes and phosphoproteins responsible for organic acid metabolism and secretion.
Project description:Light spectrum quality is an important signal for plant growth and development. We aimed to analyze the effects of different light spectra on in vitro shoot development and proteomic and polyamine (PA) profiles in shoots of Cedrela fissilis. Cotyledonary and apical nodal segments were grown under different light emitting diode (LED) lamps and a fluorescent lamp. Shoots from cotyledonary nodal segments cultured with 6-benzyladenine (BA) grown under WmBdR LED increased their length, fresh and dry matter compared to shoots grown under fluorescent light. A non-redundant protein databank generated by transcriptome sequencing and de novo assembly of C. fissilis improved, and almost doubled, protein identification compared to a Citrus sinensis databank. Using the C. fissilis protein databank, a total of 616 proteins were identified, with 23 up- and 103 downaccumulated in shoots under WmBdR LED compared to fluorescent lamp. Differential accumulation of argininosuccinate synthase protein was associated with an increase in free-Put contents and, consequently, with higher shoot elongation under WmBdR LED. Furthermore, the proteins S-adenosylmethionine synthase, which is related to PA and ethylene biosynthesis, and 1-aminocyclopropane-1-carboxylate oxidase, related to ethylene biosynthesis, were unique in shoots grown under fluorescent lamp, showing lower elongation of shoots, possibly due to ethylene production. The downaccumulation of calreticulin, heat shock proteins, plastid-lipid-associated protein, ubiquitin-conjugating enzymes, and ultraviolet-B receptor UVR8 isoform X1 could be related to better shoot length under LED. This work provides important data related to the effects of light spectrum quality on in vitro morphogenesis via modulation of specific proteins and free-Put biosynthesis.