Sequence Requirements for Cascade Function in a Type I-E CRISPR System: RNA-Seq
ABSTRACT: We measured global RNA levels using RNA-seq in cas3 E. coli cells with intact CRISPR arrays or cells with the CRISPR-I array deleted to determine if off-target events driven by endogenous spacers affect local gene expression.
Project description:We measured global RNA levels using RNA-seq in cas3+ E. coli cells with intact CRISPR arrays or cells with the CRISPR-I array deleted to determine if off-target events driven by endogenous spacers affect RNA levels.
Project description:We investigated the effect that intragenic s54 binding sites have on transcripton elongation. In an rpoN deletion we have over-expressed rpoN from a plasmid (with vector only contol) and then looked at changes in transcription elongation around intragenic s54 binding sites by RNA-seq. We are also looking at changes in transcription initiation/elongation at sites of sigma factor overlap.
Project description:The aim of the experiment was to determine differences in RNA levels between wild-type E. coli and E. coli with an hns deletion. We used RNA-seq, but to control for variation in sample preparation and sequencing, we combined each E. coli RNA sample with a spike-in Salmonella RNA sample of known concentration. Libraries constructed in duplicate from strains grown with and without Salmonella spike-in to assess absolute genome-wide changes in transcription.This allowed us to normalise RNA levels between samples during data analysis.
Project description:This experiment consists of two studies. The first where two independent biological replicates of MG1655, MG1655 thyA suhB::thyA and MG1655 thyA nusB::thyA were each grown in LB to mid-exponential phase. RNA-seq and associated data analysis were performed as described previously (Stringer et al., 2014. PMID 24272778). The second where five cultures of MG1655 thyA suhB::thyA were grown overnight from single colonies at 37 C in LB. 5 L of each overnight culture was spread on LB agar and incubated at 30 C, the non-permissive temperature for suhB mutants. One suppressor mutant colony was selected from each plate. rnc was PCR amplified from colonies using oligonucleotides JW836-JW837, and the PCR products were sequenced to identify the presence, if any, of suppressor mutations. Genomic DNA from a strain with wild-type rnc was prepared using a DNeasy Blood and Tissue Kit (Qiagen). A DNA library was prepared using a Nextera kit (Illumina). The library was sequenced (paired-end reads) using an Illumina MiSeq instrument.
Project description:Using RNAseq, the goal of the study is to investigate transcription factor regulation of virulence genes in 14028s Salmonella Typhimurium grown in SPI-1-inducing conditions. Transcription factors were cloned into pBAD24. RNA levels were compared in 14028s Salmonella Typhimurium with the transcription factor-encoding gene deleted, carrying either empty pBAD24, or pBAD24 expressing the transcription factor corresponding to the deletion. The Transcription factors analyzed are HilA, HilC, HilD, SprB, InvF, RtsA and RtsB.
Project description:This experiment contains RNA-seq data for a motile derivative of MG1655 and isogenic strains with deletions of each flagellar regulator: flhD, flhC, and fliA. All strains were grown at 37 degrees C in LB. After RNA purification, ribosomal RNA was removed using RiboZero and a library was made of remaining total RNA.
Project description:We used RNA-seq to determine the effects of AraC and arabinose on RNA levels genome-wide in S. enterica. Wild-type or delta araC mutant cells were grown in both the presence and absence of 0.2% L-arabinose. RNA-Seq libraries from two independent biological replicate samples were sequenced for (i) wild-type cells with no treatment, (ii) wild-type cells treated with arabinose, (iii) delta araC cells with no treatment, (iv) delta araC cells treated with arabinose. Comparisons were made between (i) and (ii) [analysis file name: Salmonella RNA-seq processed WT without arabinose vs WT with arabinose.xls], between (i) and (iii) [analysis file name: Salmonella RNA-Seq proessed no arabinose WT vs no arabinose delta araC.xls], and between (ii) and (iv) [analysis file name: Salmonella RNA-Seq processed arabinose WT vs arabinose delta araC.xls]. All analysis files are available from https://www.ebi.ac.uk/arrayexpress/files/E-MTAB-1901/E-MTAB-1901.additional.1.zip
Project description:modENCODE_submission_3303 This submission comes from a modENCODE project of Gary Karpen. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We aim to determine the locations of the major histone modifications across the Drosophila melanogaster genome. The modifications under study are involved in basic chromosomal functions such as DNA replication, gene expression, gene silencing, and inheritance. We will perform Chromatin ImmunoPrecipitation (ChIP) using genomic tiling arrays. We will initially assay localizations using chromatin from three cell lines and two embryonic stages, and will then extend the analysis of a subset of proteins to four additional animal tissues/stages. 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: CHIP-chip. BIOLOGICAL SOURCE: Cell Line: ML-DmBG3-c2; Tissue: CNS-derived cell-line; Developmental Stage: third instar larval stage; Genotype: y v f mal; Sex: Unknown; NUMBER OF REPLICATES: 4; EXPERIMENTAL FACTORS: Cell Line ML-DmBG3-c2; Antibody H4 (target is H4)