Project description:Clinicians need additional metrics for predicting quality of human oocytes for IVF procedures. Human polar bodies reflect the oocyte transcript profile. Quantitation of polar body mRNAs could allow for both oocyte ranking and embryo preferences in IVF applications. The transcriptome of a polar body has never been reported, in any organism. Eight total samples. There are 2 biological replicates of the following four conditions: pooled oocytes and their sister polar bodies and a single oocyte and its sister polar body.
Project description:To identify RNAs accumulating in P-bodies (cytoslic RNP granules), P-bodies were purified from HEK293 cell extracts using a Flow Activated Particle Sorting (FAPS) method. Sorted P-bodies were compared to the pre-sorted fraction to identify RNA enriched in P-bodies.
Project description:Retinoic Acid Receptors (RARs) bind RA-response elements in regulatory regions of their target genes. While canonical RAREs comprise direct repeats of the consensus 5’-RGKTCA-3’ sequence separated by 1, 2 or 5 nucleotides (DR1, DR2, DR5), we show that shortly after RA treatement of mouse embryoid bodies or F9 cells, RARs occupy a large repertoire of DR0, DR2, DR5, DR8 and IR0 elements. In vitro, RAR-RXR bind these non-canonical spacings with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half sites with DR2 and DR0 spacings. This specific half site organisation constitutes a previously unrecognised, but frequent signature of RAR binding elements and acts as an RARE. At later stages of embryoid body differentiation, RARs relocalise to a restricted repertoire of sites comprising predominantly DR5 elements. Differentiation thus involves genomic relocalisation of RARs, and a switch from DR0 and DR8 at early times to DR5 at later stages. Examination of genomic localisation of RAR in differentiating embryoid bodies.
Project description:In mesenchymal-like cell migration, cells need to polarise into a protrusive front and a retracting cell body. Apart from proteins, several mRNAs are known to be localised to cell protrusions but to what extent RNA localisation and local translation contributes toward front-back assymetry is unknown. We set out to quantify the relative translation rates of cellular proteins between protrusions and cell-body by Pulsed-SILAC proteomics, using MDA-MB-231 cells, a highly invasive breast cancer cell-line. We utilised a transwell filter based fractionation method in conjugation with pulsed-SILAC. In this method, cells are seeded on top of 3μm transwell filters to enable protrusions to form through the pores of the filters but to prevent the cell-bodies passing through due to the small size of the pores, thus resulting in separation of protrusions and cell-bodies on opposite sides of the filter, which can then be lysed and prepared separately. Prepration of protrusion and cell-body fractions from SILAC heavy vs. medium label pulsed cells then allows for reciprocal mixing and quantification of nascent proteins between protrusions and cell-body. We determined the relative local translation rates of 1150 proteins between protrusions and cell-body from two pulsed-SILAC mixes.
Project description:In mesenchymal-like cell migration, cells need to polarise into a protrusive front and a retracting cell body. To understand this process better, we set out to quantify the distribution of cellular proteins between protrusions and cell-body by proteomics, using MDA-MB-231 cells, a highly invasive breast cancer cell-line. We utilised a transwell filter based fractionation method in conjugation with SILAC proteomics. In this method, cells are seeded on top of 3μm transwell filters to enable protrusions to form through the pores of the filters but to prevent the cell-bodies passing through due to the small size of the pores, thus resulting in separation of protrusions and cell-bodies on opposite sides of the filter, which can then be lysed and prepared separately. Prepration of protrusion and cell-body fractions from heavy and light SILAC labelled cells then allows for reciprocal mixing and quantification of proteins between protrusions and cell-body. In this study, we determined the relative distribution of 3240 proteins between protrusions and cell-body from two SILAC mixes. Associated ArrayExpress data: E-MTAB-2546.
Project description:Seed lipid bodies (LB), previously viewed as paradigmatic, possess a restricted proteome and serve carbon and energy storage. Albeit present in minute quantities, LBs are widespread in post-germinative tissues, where they display vigorous movement, mediate transient organellar interactions, and shuttle enzymes, and signaling molecules. Storage as well as motility and signaling are relevant in shoot meristems of woody perennials, which ahead of winter produce buds that like seeds establish dormancy, dehydrate, express OLEOSIN genes and accumulate LBs. Unlike seeds, buds do not completely desiccate and LBs retain potential signaling functions. Here, we mapped bud LB production under dormancy-inducing conditions. We found that over an 8-week period LBs enlarged significantly. Concomitantly, three major OLEOSIN family genes were transiently upregulated at the start of bud formation, while in their wake expression of lipid biosynthesis gene DGAT1a/b and lipase gene SDP1a/b increased. In parallel, LDAPa/b, LDAP2a, and LDAP3a/b were upregulated, especially LDAP1b. All LDAPs were localized to LBs. The conclusion that OLEOSINs were replaced by LDAPs was supported by the finding that PUX10a, PUX10b and CDC498A1, involved in OLESOSIN ubiquitination and degradation, were significantly upregulated. Our proteomic analysis identified 723 putative LB proteins. These included LB-anchored Caleosin and LDIP, peripherally associated LDAP1/3 and OBL1, and numerous proteins that reflect storage, transport, organellar interaction, opportunistic cargo and contaminants. There is a significant overlap with proteins identified earlier in plasmodesmal proteomes. Collectively, the data support a model in which LBs are part of a pre-installed mechanism that serves dormancy release, survival through winter and regrowth.
Project description:Differentiation of pluripotent cells to generate lentoid bodies is important for the understanding of the lens development and investigating the processes critical for lens morphogenesis. This Study was initiated to investigate a comprehensive proteome profiling of the peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cell (iPSC)-derived lentoid bodies through mass spectrometry-based protein sequencing. Briefly, a small aliquot of blood sample was ascertained to collect PBMCs that were reprogrammed to iPSCs using the Sendai-virus delivery system. The PBMC-originated, iPSCs were differentiated into lentoid bodies employing the “fried egg” method using feeder-free conditions. The quantitative real-time PCR (qRT-PCR) confirmed the expression of lens-associated markers, which exhibited at least an order magnitude increased expression in lentoid bodies at differentiation day 25. Subsequently, the total cellular protein was extracted from lentoid bodies at day 25, digested with trypsin, fractionated into 24 fractions and subjected to an mass spectrometry-based label-free quantitative proteomics. mass spectrometry-based proteome profiling revealed 9,473 proteins in iPSC-derived lentoid bodies at differentiation day 25. In here, we report a comprehensive proteome of PBMC-originated, iPSC-derived lentoid bodies at day 25, which will help in better understanding processes critical for the development of the ocular lens.
Project description:The mRNA abundance in the wild-type or Phd2 knockout carotid body and adrenal medulla of mice was analysed by RNA-Seq. For this purpose, mice with tyrosine hydroxylase-restricted inactivation of Phd2 were generated. For the preparation of a single RNA-Seq library, RNA from 10 carotid bodies or adrenal medullas from 5 mice was pooled.
Project description:modENCODE_submission_2555 This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: RNA-seq. BIOLOGICAL SOURCE: Developmental Stage: Adult female; Genotype: wild type; Sex: Female; EXPERIMENTAL FACTORS: Strain Canton S; Tissue Female body; Replicate type: biological;
Project description:Human Primordial Germ Cell (PGC)-like cells (PGCLC) were specified in vitro from naive-like hiPS cell lines. To evaluate the first steps of PGCLC differentiation, we performed scRNA-seq (droplet-based technology, 10x) on cell suspension from embryoid body (EB) obtained after EB aggregation followed by 4 days of culture (EBd4) with cocktail of cytokines (BMP4, LIF, SCF, EGF and ROCK inhibitor) supplemented with 0.1 mM 2-mercaptoethanol and 15% KSR. In this study, two pools of embryoid bodies (EBD4_A, EBD4_B) were cultured independently for for further encapsulation and sequencing.