title: NextSeq 500 sequencing; Transcription start site identification in Bacillus subtilis
design: Transcription start site identification in Bacillus subtilis
library_construction_protocol: Duplicate cultures of  B. subtilis strain 168 ca were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen.  B. subtilis strain 168 ca was grown until mid-exponential phase in LB media with shaking at 37⁰C 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 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).
attributes.tag: Experimental Factor: replicate
attributes.value: a