Comment[ArrayExpressAccession]	E-GEOD-44582
MAGE-TAB Version	1.1								
Public Release Date	2013-04-23
Investigation Title	Processing-Independent CRISPR RNAs Limit Natural Transformation in Neisseria meningitidis								
Comment[Submitted Name]	Processing-Independent CRISPR RNAs Limit Natural Transformation in Neisseria meningitidis
Experiment Description	In bacteria and archaea, CRISPR loci confer adaptive, sequence-based immunity against viruses and plasmids. CRISPR interference is specified by CRISPR RNAs (crRNAs) that are transcribed and processed from CRISPR spacers and repeats. Pre-crRNA processing is essential for CRISPR interference in all systems studied thus far. Here we examine crRNA biogenesis and CRISPR interference in naturally competent Neisseria spp., including the human pathogen N. meningitidis. Our studies reveal a unique crRNA maturation pathway in which crRNA transcription is driven by promoters that are embedded within each repeat, yielding crRNA 5’ ends are not formed by processing. Although crRNA 3’ end formation occurs through RNase III cleavage of a pre-crRNA/tracrRNA duplex, as in other Type II CRISPR systems, this processing event is dispensable for interference. The meningococcal pathway is the most streamlined CRISPR/cas system characterized to date. Endogenous CRISPR spacers frequently target genomic sequences of other Neisseria strains and so limit natural transformation, which is the primary source of genetic variation that contributes to immune evasion, antibiotic resistance, and virulence in N. meningitidis. dRNA-seq approach for RNA samples from cultures of N. lactamica 020-06, harvested at mid-log. Two cDNA libraries from total RNA were prepared to distinguish between transcripts with either primary orprocessed 5’ ends: one library is generated from untreated RNA, whereas the other is treated with terminator exonuclease (TEX),								
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl							
Person Last Name	Förstner	Zhang	Heidrich	Joseph	Gunderson	Seifert	Schoen	Vogel	Sontheimer
Person First Name	Konrad	Yan	Nadja	Biju	Carl	H	Christoph	Jörg	Erik
Person Mid Initials	U.					S			J
Person Email	konrad.foerstner@uni-wuerzburg.de								
Person Affiliation	University of Würzburg								
Person Address	Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Str. 2/D15, Würzburg, Bavaria, Germany								
Person Roles	submitter								
Protocol Name	P-GSE44582-2	P-GSE44582-1							
Protocol Description	Demultiplexing FastQ quality trimming using FastX and a cut-off value of 20 Fastq to fasta conversion using FastX Removal of polyA-tails from Fasta sequences Size filtering: discarding reads shorter than 12 nt Read mapping using segemehl version 0.9.4 Coverage calculation and normalization Genome_build: NC_014752.1 Supplementary_files_format_and_content: Wiggle	Frozen cell pellets from liquid cultures were lysed, and total RNA was extracted from the lysates using the hot-phenol method described previously (Blomberg et al., 1990). For depletion of processed transcripts, total RNA was freed of residual genomic DNA by DNase I treatment, and equal amounts of Neisseria RNA were incubated with Terminator 5’-phosphate-dependent exonuclease (TEX) (Epicentre) or in buffer alone as previously described (Sharma et al., 2010). Libraries for Solexa sequencing (HiSeq) of cDNA were constructed by vertis Biotechnology AG, Germany (http://www.vertis-biotech.com/), as described previously for eukaryotic microRNA (Berezikov et al., 2006) but omitting the RNA size-fractionation step prior to cDNA synthesis.							
Protocol Type	normalization data transformation protocol	nucleic acid library construction protocol							
Comment[SecondaryAccession]	GSE44582								
Comment[GEOReleaseDate]	2013-04-23								
Comment[ArrayExpressSubmissionDate]	2013-02-22								
Comment[GEOLastUpdateDate]	2013-04-24								
Comment[AEExperimentType]	RNA-seq of coding RNA								
Comment[SecondaryAccession]	SRP018813								
Comment[SequenceDataURI]	http://www.ebi.ac.uk/ena/data/view/SRR749074-SRR749075								
SDRF File	E-GEOD-44582.sdrf.txt