Unknown,Transcriptomics,Genomics,Proteomics

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RIP-seq identification of RNAs enriched on RNA polymerase and the primary sigma factor (sigma A) in Bacillus subtilis

ABSTRACT: 6S RNA is a small RNA with specific secondary structure that associates with the complex of RNA polymerase (RNAP) and the primary sigma factor in majority of bacteria. Bacillus subtilis has two 6S RNAs. To compare both 6S RNAs and to identify RNAs that might have a similar functions, we sequenced RNAs that co-immunoprecipitated with RNAP or the primary sigma factor (sigma A). We also sequenced total RNA isolated from the lysate (input sample). We expect that putative 6S RNA-like molecules are enriched in RNA polymerase or sigma A samples compared to the inputs. We performed the experiment both in exponential and stationary phase of growth.

INSTRUMENT(S): NextSeq 550

ORGANISM(S): Bacillus subtilis subsp. subtilis str. 168

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PROVIDER: E-MTAB-11693 | biostudies-arrayexpress |

SECONDARY ACCESSION(S): E-MTAB-11692

REPOSITORIES: biostudies-arrayexpress

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Project description:We determined genome-wide nucleosome occupancy in mouse embryonic stem cells and their neural progenitor and embryonic fibroblast counterparts to assess features associated with nucleosome positioning during lineage commitment. Cell type and protein specific binding preferences of transcription factors to sites with either low (e.g. Myc, Klf4, Zfx) or high (e.g. Nanog, Oct4 and Sox2) nucleosome occupancy as well as complex patterns for CTCF were identified. Nucleosome depleted regions around transcription start and termination sites were broad and more pronounced for active genes, with distinct patterns for promoters classified according to their CpG-content or histone methylation marks. Throughout the genome nucleosome occupancy was dependent on the presence of certain histone methylation or acetylation modifications. In addition, the average nucleosome-repeat length increased during differentiation by 5-7 base pairs, with local variations for specific genomic regions. Our results reveal regulatory mechanisms of cell differentiation acting through nucleosome repositioning. For chromatin immunoprecipitation, for each sample, 1 x 106 cells were cross-linked with 1% PFA and cell nuclei were prepared using a swelling buffer (25 mM Hepes pH 7.8, 1 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT). Chromatin was sheared to mononucleosomal fragments. After IgG preclearance the sheared chromatin was incubated with 4 Âµg of either a H3K9ac (Abcam, ab4441), a H3K27ac (Abcam, ab4729), or a H3K9me3 (Abcam ab8898) antibody over night. After washes with sonication (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% N-lauroylsarcosine, 0.1% Na-deoxycholate), high-salt- (50 mM Hepes pH 7.9, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), lithium- (20 mM Tris-HCl pH 8.0, 1mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate) and 10 mM Tris-HCl, chromatin was eluted from the protein G magnetic beads and the crosslink was reversed over night. After RNase A and proteinase K digestion, the DNA was purified and subsequently cloned into a multiplexed Illumina library according to standard protocols. Sequenced 50 bp reads were mapped with bowtie and subsequently clustered with MACS 55 implemented in the Genomatix software suite (Genomatix, Munich, Germany) using a p-value of 10-5.

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RIP-seq identification of RNAs enriched on RNA polymerase and the primary sigma factor (sigma A) in Bacillus subtilis

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