2025-10-30
ENA first public
ERC000011
ENA-CHECKLIST
Institute for Microbiology and Infection, University of Birmingham
INSDC center name
public
INSDC status
ERS24142596
SRA accession
E-MTAB-15087:Cappable_seq_a
Submitter Id
ArrayExpress
broker name
not collected
collection date
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).
description
not collected
geographic location (country and/or sea)
M22+ media. 30 degrees Celsius
growth condition
not applicable
isolate
Cereibacter sphaeroides
organism
Cereibacter sphaeroides
organism
biological replicate a
replicate
N/A
response to treatment
Cereibacter sphaeroides
scientific_name
Rhodobacter sphaeroides 2.4.1
strain
Cappable_seq_a
title