Mycobacterium tuberculosis H37Rv
Protocols: Mycobacterium tuberculosis H37Rv (SysteMTb) was grown in Middlebrook 7H9 medium supplemented with 0.4% glycerol, 0.085% NaCl, 0.5% BSA and 0.05% Tyloxapol in roller bottle culture (2 rpm at 37?C). For starvation experiments, exponentially growing bacteria were washed, resuspended in PBS supplemented with 0.025% Tyloxapol, and maintained in roller bottle culture for a further 24 hours RNA was isolated from triplicate exponentially growing and PBS-washed cultures using the FastRNA Pro blue kit from QBiogene/MP Bio according to manufactures instructions. All RNA samples were treated with Turbo DNase free (Ambion). For the synthesis of whole transcriptome cDNA, RNA was fragmented with ultrasound (4 pulses of 30 s at 4oC), then treated with Antarctic phosphatase and re-phosphorylated with polynucleotide kinase (PNK). Fragmented RNA was then poly(A)-tailed using poly(A) polymerase and a RNA adapter was ligated to the 5'-phosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. The resulting cDNA was PCR-amplified to about 20-30 ng/?l using a high fidelity DNA polymerase. For TSS mapping, RNA was enriched for primary transcripts by incubating fragmented, PNK-treated samples with Terminator exonuclease (TEX, Epicentre) which specifically degrades RNA species carrying a 5' monophosphate (5'P). Exonuclease-resistant RNA species (primary transcripts with 5'PPP) were poly(A)-tailed using poly(A) polymerase, followed by treatment with tobacco acid pyrophosphatase (TAP, Epicentre) to degrade 5'PPP to 5'P RNA
ENA-FIRST-PUBLIC
2013-11-25
ENA-LAST-UPDATE
2013-04-17
External Id
SAMEA2064714
INSDC center alias
MRC National Institute for Medical Research
INSDC center name
MRC National Institute for Medical Research
INSDC first public
2013-11-25T17:03:13Z
INSDC last update
2013-04-17T14:30:30Z
INSDC status
public
Submitter Id
E-MTAB-1616:Starv_R1_WT
broker name
ArrayExpress
growth condition
Starved cultures
sample name
E-MTAB-1616:Starv_R1_WT
strain
H37Rv
time
24 hour
type-material
type strain of Mycobacterium tuberculosis