7002
Mycolicibacterium smegmatis MC2 155
Protocols: Msm SMR5 wild-type and Msm SMR5 ΔpafBC cells were grown in triplicates in Middlebrook 7H9 medium as 50 ml shaking cultures to an OD600 between 0.4 to 0.7 at 37 °C. The cultures were incubated for another 1.5 h at 37 °C in presence or absence of 80 ng/ml MMC (Roche #000000010107409001) to induce DNA damage stress. The cultures were incubated for another 1.5 h at 37 °C. Cells were harvested by centrifugation (3200 g, 10 min, 25 °C) from 10 ml culture. Trimming (CLC Genomics Workbench Version 10.0.1; Trim Sequences v1.13) LX_illumina_adapters_for3p Sequence = AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT Strand = Minus Action = Remove adapter Scores = Mismatch: 2, Gapcost: 3, Cutoff: 10, Cutoff at end: 4 Trim adapter list = LX_illumina_adapters_for3p Ambiguous trim = No Quality trim = Yes Quality limit = 0.05 Use colorspace = No Create report = Yes Also search on reversed sequence = No Save discarded sequences = Yes Remove 5' terminal nucleotides = Yes Number of 5' terminal nucleotides = 9 Minimum number of nucleotides in reads = 15 Discard short reads = Yes Remove 3' terminal nucleotides = No Discard long reads = No Save broken pairs = No If not stated otherwise, the manufacturer's protocol applies. Total RNA was isolated using the SPLIT RNA Extraction Kit (Lexogen #008) after a modified protocol: Cells were resuspended in 600 μl ice-cold buffer IB, transferred on ice to 2 ml screw cap tubes containing 500 mg to 700 mg 0.15 mm zirconium oxide beads (NextAdvance #ZrOB015) and 3 μl Reagent DX (Qiagen #19088). Cell lysis was performed by bead beating for 30 s at 5000 rpm (Precellys Minilys). Insoluble material was removed by centrifugation at 19000 g, 10 min, 4 °C. 400 μl cleared lysate were used for the acidic phenol extraction step according to the kit's protocol while replacing chloroform with 1-bromo-3-chloropropane. A second extraction step was performed using 1 ml 1-bromo-3-chloropropane to remove trace amounts of phenol. The extracted samples were further processed according to the kit's protocol. Total RNA was then treated with the TURBO DNA-free Kit (Thermo Scientific #AM1907) to remove residual genomic DNA and afterwards purified using RNAClean XP beads (Agencourt). RNA concentrations were determined using Qubit RNA quantification assays (Thermo Scientific). Sample integrity was monitored with a TapeStation 2200 (Agilent) instrument. Presence of genomic DNA was assayed by PCR for amplification of a 245 bp fragment of the rpoB coding sequence (primers: rpoB-fw GTTCGCCTGGCGTGTACTT and rpoB-rt CAGCGTGCCCATCATGATCT). For storage, samples were kept at -80 °C. If not stated otherwise, the manufacturer's protocol applies. Ribosmal RNA was depleted with the Ribo-Zero Gram-Positive Bacteria Kit (illumina #MRZGP126) and the depleted RNA was purified using RNAClean XP beads. Sequencing libraries were prepared using the SENSE Total RNA-Seq Library Prep Kit (Lexogen #009) with 15 ng rRNA-depleted total RNA as input, an incubation time of 1.5 h for the reverse transcription step, the PE100 size selection parameters according to the manual, and 16 amplification cycles in the final PCR step. RNA concentrations were determined using Qubit RNA quantification assays (Thermo Scientific). Sample integrity was monitored with a TapeStation 2200 (Agilent) instrument. Presence of genomic DNA was assayed by PCR for amplification of a 245 bp fragment of the rpoB coding sequence (primers: rpoB-fw GTTCGCCTGGCGTGTACTT and rpoB-rt CAGCGTGCCCATCATGATCT). For storage, samples were kept at -80 °C.
ENA first public
2018-06-17
ENA last update
2018-02-14
External Id
SAMEA104590942
INSDC center alias
ETH Zurich Institute of Molecular Biology and Biophysics
INSDC center name
ETH Zurich Institute of Molecular Biology and Biophysics
INSDC first public
2018-06-17T17:02:46Z
INSDC last update
2018-02-14T15:16:30Z
INSDC status
public
Submitter Id
E-MTAB-6497:7002
broker name
ArrayExpress
genotype
delta pafBC
growth condition
Middlebrook 7H9 medium
sample name
E-MTAB-6497:7002