biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsDavid ForrestIllumina NovaSeq 6000NextSeq 500RNA-seq of total RNACereibacter sphaeroidesCereibacter sphaeroideshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15087Sequencing datasets produced during the investigation into the function of the CarD transcription factor in Rhodobacter sphaeroides. Sequencing datasets and in vitro biochemical assay reveal CarD is dependant on negative DNA supercoiling for activation of transcription. In this experiment, 4 sets of data was produced: RNA-seq - determining gene expression levels Cappable-seq - determing transcription start sites ChIP-seq - determing CarD and Sigma factor binding sites Psora-seq - determining global levels of DNA supercoilingbiostudies-arrayexpressLibrary Construction - Cappable-seq: Library preparation and sequencing was done by Vertis Technologie AG according to the protocol described by Ettwiller et al (2). Briefly, 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) using Vaccinia capping enzyme (New England Biolabs). Biotinylated RNA was captured and eluted from streptavidin beads to obtain 5' fragments of primary transcripts. These transcripts were poly(A) tailed with poly(A) polymerase before conversion of the 5' CAP moiety to a 5' monophosphate using CAP-clip Acid pyrophosphatase (Cellscript). An RNA adapter was ligated to the 5' monophosphate and cDNA synthesis was done with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. cDNAs were amplified by PCR to a final concentration of 10-20 ng µl-1. Full length cDNAs were fragmented and immobilised with streptavidin magnetic beads for blunting and ligation of the 3' Illumina sequencing adapter. The immobilised cDNA fragments were amplified via PCR. The sample libraries were mixed in equimolar amounts 200-500 bp fragments were purified from an agarose gel after electrophoresis. 2 - Ettwiller, L., Buswell, J., Yigit, E. & Schildkraut, I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics 17, 199 (2016).Library Construction - DNA was sent to Gene wiz (Azenta) for sequencing, with samples treated as ChIP-seq samplesGrowth Protocol - For each biological replicate, a single colony was inoculated into 10 ml M22+ media and grown at 30 degrees Celsius with vigorous shaking for 2-3 days. This 10 ml culture was used to start a larger culture, typically 100 - 200 ml. The larger culture had a starting OD600 of 0.05, and was grown throughout the day at 30 degrees Celsius with shaking until exponential phase, approx 0.5 OD600. At which point novobiocin was added for 20 minutes if required.Sample Collection - All cells were harvested by high speed centrifugation (approx 5000 xG). Pellets were flash froze in liquid nitrogen before storage at -80 oC.Sample Treatment - When stated, novobiocin was added to a exponentially growing cells (OD600 0.5) to a final concentation of 2 μg/mlNucleic Acid Extraction - Experiments were done as described by Visser et al with the following changes 28. CTAB purified genomic DNA was resuspended in 225 μl of water. Genomic DNA was sheared to a size of 400 – 500 bp in a Biorupter (Diaganode). 50 μl of sheared genomic DNA was kept as an input sample and the remainder was immobilised on streptavidin T1 dynabeads (ThermoFisher). After washing, and reverse crosslinking as per Visser et al, eluted DNA was precipitated and resuspended in 20 μl water Visser, B. J., Sharma, S., Chen, P. J., McMullin, A. B., Bates, M. L. & Bates, D. Psoralen mapping reveals a bacterial genome supercoiling landscape dominated by transcription. Nucleic Acids Res. 50, 4436–4449 (2022)Nucleic Acid Extraction - Total RNA was isolated as described previously (1) with the exception that RNA concentration and quality was determined on an Agilent 2200 Tapestation. 1 - Forrest, D., James, K., Yuzenkova, Y. & Zenkin, N. Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase. Nat. Commun. 8, 15774 (2017)Library Construction - Library construction for strand-specific RNA-seq was performed Gene Wiz (Azenta)Library Construction - Sequencing libraries were prepared using the NEB next Ultra II kit (New England Biolabs). Library concentrations were determined by qPCR using a NEB next library quantification kit (New England Biolabs).Sequencing - Libraries were sent to Gene Wiz (Azenta) and sequenced using an Illumina NovaSeq instrument, generating paired end libraries 150 bp in length.Sequencing - Strand-specific RNAseq libraries were sequenced by Gene Wiz (Azenta) on a NovaSeq 6000, generating paired end 150 bp reads.Nucleic Acid Extraction - Exponentially growing R. sphaeroides, with or without 2 μg/ml novobiocin, were crosslinked with 1% (v/v) formaldehyde for 20 minutes. The reaction was quenched with 0.5 M glycine and cells were resuspended in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 % (v/v) Triton X-100, 0.1% (v/v) sodium deoxycholate and 1 protease inhibitor cocktail tablet per 50 ml) with 2 mg ml-1 lysozyme. After sonication, 20 μl of chromatin was kept as an input control. Approximately 150 μg of chromatin was immunoprecipitated by the addition of anti-CarD antibodies (a gift from Wilma Ross, University of Wisconsin-Madison) or anti-σ antibodies (Biolegend). Protein-DNA complexes were immunoprecipitated with a 1:1 mix of Protein A/G Dynabeads (Thermo Fisher). Before eluting complexes, beads were washed with lysis buffer, high salt (500 mM NaCl) lysis buffer, wash buffer (20 mM Tris pH 8, 250 mM LiCl, 0.5 % (v/v) NP-40, 0.5 % (v/v) sodium deoxycholate and 1 mM EDTA) and TE. Elution, protein degradation (1 hour incubation with proteinase K at 42 °C) and reverse crosslinking (4 hours at 65 °C) was done in elution buffer (50 mM Tris pH 7.5, 10 mM EDTA and 1 % (v/v) SDS). DNA purified from eluate using a Qiaquick PCR clean up kit (Qiagen).Sequencing - Paired end 150 bp reads were generated by sequencing libraries on an Illumina NovaseqSequencing - The libraries were sequenced on an Illumina Nextseq 500 system with a read length of 75 bpOrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesDavid ForrestfalseCarD links DNA supercoiling to gene regulation in Rhodobacter sphaeroidesSequencing datasets produced during the investigation into the function of the CarD transcription factor in Rhodobacter sphaeroides. Sequencing datasets and in vitro biochemical assay reveal CarD is dependant on negative DNA supercoiling for activation of transcription. In this experiment, 4 sets of data was produced: RNA-seq - determining gene expression levels Cappable-seq - determing transcription start sites ChIP-seq - determing CarD and Sigma factor binding sites Psora-seq - determining global levels of DNA supercoiling2025-10-16T00:00:00Z2025-10-16T01:02:33.676Z2025-04-24T15:57:20.773ZE-MTAB-15087ERP171952EFO_0002944EFO_0004170EFO_0009653EFO_0003789EFO_0005518EFO_0004184EFO_0003969