ABSTRACT: Over-night cultures grown from three single colonies were diluted 1:100 in LB and grown with shaking at 37 °C for 6h. Bacteria were pelleted by centrifugation and fast-freezed in the presence of 0.9 mg/ml lysosyme in TE. Frozen cells were thawed and re-frozen twice in 37 °C and liquid nitrogen, respectively. Total RNA was extracted using TRI-reagent and used for construction of RNA-seq libraries according to the RNAtag-seq method. Libraries were paired-end sequenced using Illumina NextSeq 500 platform.
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Escherichia coli str. K-12 substr. MG1655
Project description:SGH10 bacteria were grown under DMEM (Dulbecco's Modified Eagle Medium) and LB(Lysogeny broth). Bacteria were harvested by centrifugation, the pellet was resuspended in TE buffer, mixed with lysosyme (SigmaAldrich) in a final concentration of 0.9 mg/ml, and immediately frozen in liquid nitrogen. The frozen pellet was subjected to two cycles of thaw and freeze at 37°C and in liquid nitrogen, respectively, and RNA was extracted using Tri-Reagent (Sigma-Aldrich). RNA-seq libraries were constructed by the RNAtag-seq method. RNA-seq libraries were sequenced by single end sequencing using Nextseq 500 Sequencer (Illumina). sequence fragments were mapped to SGH10 which contains a chromosome NZ_CP025080.1 and a plasmid NZ_CP025081.1. For each growth condition, three biological repeats were done.
Project description:RIL-seq experiment of EPEC hfq-flag mutant, in activating- conditions:growth on DMEM at 37°C to mid exponential growth phase (e.g., OD600=0.3). In these conditions EPEC strongly expresses its major virulence components, T3SS and BFP, mimicking infection. Non-activating conditions: overnight growth of static culture on LB medium at 37°C where virulence factors are not expressed. RIL-seq experiments are designed to reveal the interactions of sRNA and their targets.
Project description:RIL-seq experiment allows identification of pairs of interacting RNA molecules while bound to the Hfq protein. SGH10 bacteria were grown under DMEM (Dulbecco's Modified Eagle Medium) and LB (Lysogeny broth) conditions. RIL-seq was performed as previously described in Melamed et al. 2018 with adaptation of the computational analysis to the SGH10. It was applied to three biological replicates, for each growth condition. Libraries were sequenced by paired-end sequencing using Nextseq 500 Sequencer (Illumina). sequence fragments were mapped to SGH10 which contains a chromosome NZ_CP025080.1 and a plasmid NZ_CP025081.1. RIL-seq derived sequence fragments that mapped to the same locus or to two different loci were defined as single and chimeric fragments respectively. Chimeric fragments that appeared statistically significantly more than expected at random (S-chimeras) were considered as representing putative interacting RNAs.
Project description:In this study we developed TRS, a computational approach to determine the 3’ termini of transcripts from total RNA data generated by the RNAtag-seq protocol. To show the applicability of our approach, we applied our algorithm to sequencing data generated by the RNAtag-seq and the term-seq protocols. In term-seq, a 3’ adaptor is ligated to the RNA 3' termini prior to the RNA fragmentation, and thus, the sequences adjacent to the adapter sequence in the library reads represent original 3' termini that were present in the RNA sample. The RNAtag-seq protocol involves ligation of a 3’ adaptor after the RNA fragmentation and thus, the sequences adjacent to the adapter sequence in the library reads represent genuine 3’ termini that were present in the RNA sample and artificial 3’ termini generated by the RNA fragmentation. We show that our computational approach can reliably identify genuine 3’ termini from RNAtag-seq data by comparing the set of identified 3’ termini based on the RNAtag-seq and the term-seq protocols.
Project description:For expression analysis of wild-type V. cholerae, hapR, and rpoS deletion mutants in mid-exponential or stationary phase, the strains were grown to either OD600 of 0.3 or for 11 h in LB media at 37 0C, and bacteria from 2-ml culture were quickly pelleted, resuspended in Trizol reagent (GIBCO/BRL, San Diego, California, United States), and frozen on dry ice. RNA was isolated from the Trizol agent, treated with DNaseI (Ambion, Austin, Texas, United States), and cleaned by using the RNeasy kit (Qiagen, Valencia, California, United States). Labeling of cDNA and microarray hybridizations were performed as described [Yiliz et al. 2001, Mol. Micro. 53: 497-515]. Microarrays were scanned with a GenePix 400A instrument (Axon Instruments), using the GENEPIX 5.0 software. At least four microarray experiments were performed for each of two biological replicates for the tested strains. Gene expression of V. cholerae, rpoS, and hapR deletion mutants in stationary phase LB cultures was analyzed and compared to the wild-type parent under identical conditions. Gene expression of the wild-type parent during stationary phase after 11 h growth in LB was analyzed using RNA from an exponentially growing culture as a reference. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
Project description:The RNA content of the CsrA foci. EPEC (E2348/69 (strain O127:H6)) expressing CsrA-FLAGx3-GFP or wild-type EPEC expressing untagged CsrA (negative control) were grown to OD 0.6 in DMEM and subjected to the foci-enrichment protocol, followed by RNA extraction and library preparation. Six libraries were prepared, containing three biological repeats of csrA-3 x flag-gfp and three repeats of wt.
Project description:Global gene expression was monitored in long-term stationary phase (LSP) cells of E. coli K12 MG1655 and compared with stationary phase (SP) cells that were sub-cultured without prolonged delay to get an insight into the survival strategies of LSP cells. The experiments were carried out using both LB medium and LB supplemented with 10% of glycerol. In both the media the LSP cells showed decreased growth rate compared to SP cells. DNA microarray analysis of LSP cells in both the media resulted in the up- and down-regulation of several genes in LSP cells compared to their respective SP cells in the corresponding media. In LSP cells grown in LB 204 genes whereas cells grown in LB plus glycerol 321 genes were differentially regulated compared to the SP cells. Comparison of these differentially regulated genes indicated that irrespective of the medium used for growth in LSP cells expression of 95 genes (22 genes up-regulated and 73 down-regulated) were differentially regulated. These 95 genes could be associated with LSP status of the cells and are likely to influence survival and growth characteristics of LSP cells. This is indeed so since the up- and down-regulated genes include genes that protect E. coli LSP cells from stationary phase stress and genes that would help to recover from stress when transferred into fresh medium. The growth phenotype in LSP cells could be attributed to up-regulation of genes coding for insertion sequences that confer beneficial effects during starvation, genes coding for putative transposases and simultaneous down-regulation of genes coding for ribosomal protein synthesis, transport-related genes, non-coding RNA genes and metabolic genes. As yet we still do not know the role of several unknown genes and genes coding for hypothetical proteins which are either up- or down-regulated in LSP cells compared to SP cells.
Project description:Technical replicate testing was performed to determine the measurement precision of each analyte. Peripheral blood mononuculear cells were processed and frozen down from blood samples collected from healthy control subjects (n=3) or subjects with Systemic Lupus Erythematosus (n=3). Three separate PBMC aliquots per subject were then independently thawed and sorted by flow into B cell, CD4+ T cell, CD8+ T cell, and monocyte populations. Total RNA was isolated from the sorted cell populations and then RNAseq libraries were prepared.
Project description:Preparation of AMDs. 125 μM Ac4ManNAz were incubated with Raw 264.7 (~ 107 cells/dish) for 24, 48, and 72 h, followed by gentle washing with PBS or complete medium to produce Raw 264.7-Ac4ManNAz. Subsequently, Raw 264.7-Ac4ManNAz (~ 107 cells) were resuspended in PBS containing Alkynyl-Ce6(0.1 μM), ascorbic acid (0.2 μM) and CuSO4 (0.2 μM) and incubated for 1h at 37 °C. Then centrifuged (1000 rpm, 3 min) and washed 3 times with PBS to obtain precursor of AMDs. precursor of AMDs (~ 107 cells/tube, 1.0 ml) was immersed in liquid nitrogen, 24 h later, the frozen tubes were thawed at 37°C, centrifuged at 1500 rpm for 3 min, washed and resuspended by PBS to generate the AMDs (5 x 106 units/tube, 1.0 ml).
Project description:Aim to characterize the cervical transcriptome of four European ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen.