Cappable_seq_b
Cereibacter sphaeroides
Protocols: All cells were harvested by high speed centrifugation (approx 5000 xG). Pellets were flash froze in liquid nitrogen before storage at -80 oC. 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.  When stated, novobiocin was added to a exponentially growing cells (OD600 0.5) to a final concentation of 2 μg/ml 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) 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).
ENA first public
2025-10-30
INSDC center name
Institute for Microbiology and Infection, University of Birmingham
INSDC status
public
Submitter Id
E-MTAB-15087:Cappable_seq_b
broker name
ArrayExpress
collection date
not collected
geographic location (country and/or sea)
not collected
growth condition
M22+ media. 30 degrees Celsius
isolate
not applicable
replicate
biological replicate b
response to treatment
N/A
sample name
E-MTAB-15087:Cappable_seq_b
scientific_name
Cereibacter sphaeroides
strain
Rhodobacter sphaeroides 2.4.1