Project description:The king scallop Pecten maximus is a high valuable species of great interest in Europe for both fishery and aquaculture. However, hatchery production is a relatively new industry and it is still underdeveloped. Major hurdles are spawning control and gamete quality. In the present study, a total of 14 scallops were sampled in the bay of Brest (Brittany, France) to compare transcriptomic profiles of mature oocytes collected by spawning induction or by stripping. To reach such a goal, a microarray analysis was performed by using a custom 8x60K oligonucleotide microarray
Project description:Patients affected by type 1 diabetes are recruited in the departments of Diabetology and Healthy Volunteers (HV) are selected based on internal records in the same hospital. Total RNA from whole blood has been extracted following a two-step procedure. First, RNA from blood collected on PAX-Gene tubes has been extracted using Maxwell 16 LEV simplyRNA blood kit (Promega) following manufacturer recommendations and second b-globin, dominant RNA from red blood cells, has been removed using the GLOBINclear kit (Ambion) on extracted RNA. RNA sequencing has been performed from using the TruSeq Stranded mRNA preparation kit (Illumina) on 500 ng b-globin depleted RNA with a RNA Integrity Number > 8 (measured on Bioanalyzer following manufacturer recommendations), and then sequenced following a pair-end 2x75 bp protocol on NextSeq 500 or HiSeq 4000 (Illumina) at LIGAN Equipex (Lille, France).
Project description:ngs2013_07_pcapsici-effecaps-phytophthora capsici-The analysed RNAseq concerned the oomycete Phytophthora capsici in growth on pepper plants. 1/ How the adaptation of a pathogen to a host depends on its gene expression? 2/ How the plant host impacts the expression of pathogen genes at the very beginning of infection?-Two isolates of Phytophthora capsici were used: the Pc107 isolate (called A for adapted to pepper from INRAE GAFL Avignon), and the Pc273 isolate (N for non-adapted to pepper, collected on pumpkin in the USA). Two accessions of pepper (Capsicum annuum L., the host) were used: Yolo Wonder (YW, PM0031), susceptible (S) to Phytophthora capsici, and Criollo de Morelos 334 (CM334, PM0702), partially resistant (R). Inoculations were performed, as described in Lefebvre and Palloix (1996), by putting on the wounded stem a plug of mycelium. Inoculated plants were transferred to a growth chamber at 24°C/22°C temperature on a 12h/12h light/dark cycle. At 24 hours-post-inoculation, 12 total RNA samples were extracted from inoculated plants for the 4 host-isolate interactions: R_A, S_A, R_N and S_N. Each sample consisted of six pooled stem fragments. The stem fragments are the 5-mm region immediately under the visible stem necrosis. Samples were flash-frozen in liquid-nitrogen and stored at -80ºC. They were ground in liquid nitrogen with a cold mortar and pestle. Total RNA was extracted using QIAGEN Rneasy Plant Mini Kit. RNA-seq libraries were constructed at IPS2 POPS platform (France) by TruSeq_Stranded_mRNA_SamplePrep_Guide_15031047_D protocol (Illumina®, California, USA). Sequencing was conducted on an Illumina Hiseq2000 hosted by Genoscope (Evry, France). The RNA-seq samples have been sequenced in paired-end (PE) with a sizing of 260 bp and a read length of 100 bases, lane repartition and barcoding giving approximately 35 million of paired-end reads per sample.
Project description:To dissect how diurnal rhythms affect key functions such as transcription or chromatin remodeling, we quantified the temporal nuclear accumulation of proteins and phosphoproteins from mouse liver by SILAC-based MS. Protein extracts from isotope labelled mice liver nuclei were used as a reference and mixed with extracts from animals collected every 3h for 45h total. Total protein levels were analysed together with phosphopeptides after enrichment (see separate dataset for phospho data).
Project description:For determination of reactivity with rDer p 21 and Der p 21 in extracts, a serially diluted rDer p 21 from 100 to 0.00305 µg/mL and 6 different HDM extracts diluted 16 times from their stock concentrations were printed. Human serum albumin, rMBP, HRP and PBS were printed as negative controls. Each allergen and control were printed as a single droplet (~450 pL/drop) in three replicates onto 2D-Epoxy glass slides (PolyAn GmbH, Germany) using sciFLEXARRAYER SX microarray printer (SCIENION GmbH, Germany). The slides were blocked with PBS-T containing 2% BSA (blocking buffer) for 30 min at RT. After that, the slides were incubated for 2 h with MAbs 0.2 µg/mL, diluted with blocking buffer, 80 µL/well). Then the slides were incubated for 30 min with the goat anti-mouse IgG Fc Alexa Fluor® 647 (SouthernBiotech, USA) (1 μg/mL, diluted with blocking buffer, 80 μL/well). The slides were scanned using InnoScan 710 AL microarray scanner (Innopsys, France). The images were analyzed with MAPIX software (Innopsys, France). The average mean ± standart deviation value of each allergen and control was calculated using spots’ median fluorescence intensity (MFI) values
Project description:In this study, we systematically identified the RNAs associated with a selective sample of 40 of ~600 yeast RNA-binding proteins (RBPs). To identify RNAs associated with each putative RBP, C-terminal tandem affinity purification (TAP)-tagged proteins, expressed under control of their native promoters, were affinity purified from whole cell extracts of cultures grown to mid-log phase in rich medium [1-3]. Extracts were incubated with immunoglobulin G (IgG) agarose beads, washed, and ribonuclear protein complexes were eluted by tobacco etch virus (TEV) protease treatment (Text S2). We performed two to four independent isolations with each tagged strain. As controls, we performed 13 immunoaffinity purifications (IPs) of untagged strains to identify and exclude potential false-positive RNA targets. We purified total RNA from the whole-cell extracts and TEV-purified fractions, reverse transcribed with an amino-allyl-dUTP/dNTP mix, coupled the purified cDNA to Cy3 and Cy5 dyes, respectively, mixed the two differentially labeled cDNA pools, and then hybridized them to DNA microarrays (Datasets S1-S4). An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Genotype: strain expressing tagged version of RNA-BP Keywords: all_pairs
Project description:Non-targeted LC-MS/MS analysis of PPL solid phase extracted dissolved organic matter (DOM) from TARA/TREC Expedition Leg 1, collected in the coastal Atlantic between France to Netherlands in Spring 2023.
Project description:Leucocytes-decontaminated platelet-rich plasma (PRP) from 15 healthy volunteers was collected and activated with collagen, or Thrombin Activator Receptor Peptide (TRAP). Total protein extracts (75 microg protein/sample), coming from two biological replicates, were precipitated using 2-D Clean Up Kit (Amersham Bioscience). Total protein extracts were precipitated and dissolved into 20 microl of iTRAQ dissolution buffer. Protein alkylation, trypsin digestion and labeling of the resulting peptides were performed according to manufacturer’s instructions (Applied Biosystems) followed by strong cation exchange fractionation and LC-MS/MS analysis of the peptides as previously described (Lietzén et al, PLoS Pathogens). Protein identification and relative quantitation were performed with Paragon search algorithm using ProteinPilot 4 interface (Applied Biosystems). Database searches were performed against human protein sequences in UniProt database (version 120813 with 20231 human entries). The search criteria were: cysteine alkylation with MMTS, trypsin digestion, biological modifications allowed, thorough search and detected protein threshold of 95% confidence (Paragon Unused ProtScore > 1.3). Additionally, automatic bias correction was used to correct for uneven protein loading.
Project description:E. coli O157:H7 global gene expression profile is altered during growth in the presence of Hep-2 cells, relative to the absence of cells; We used Affymetrix E. coli 2.0 genechips to determine gene expression changes Experiment Overall Design: E. coli O157:H7 total RNA extracts were collected after a 6 hour regrowth period in either the presence or absence of Hep-2 cells. E. coli O157:H7 total RNA extracts were collected after a 6 hour regrowth period in minimal media with 5% CO2.