Protocols: 1 mL culture was added to 250 μL pure ethanol supplemented with 5 % phenol, flash-frozen in liquid nitrogen and stored at −80°C . Synechocystis strains were cultivated in BG11 medium with appropriate antibiotics at 30°C and aeration with 0.5% CO2 in all experiments. For the endpoint measurements cultures were grown in Erlenmeyer flask in an Infors HT multitron chamber, shaken at 150 rpm, and light intensity was 80 umol/m2/sec. For the time series 1 L culture was grown in a Lambda Photobioreactor ca. 90 umol/m2/sec per OD (manually adjusted to OD) with pH kept stable at pH 8. Inducers for gyrase knock-down and topoisomerase I overexpression were added at inoculation after pre-culturing without inducer. For the time series experiment the inducer was added at time point t=0 h, 1 h after SAMPLE_10 was taken. Frozen samples were centrifuged ( 10 min , 4°C , maximum speed). The pellet was resuspended in 1 mL PGTX and incubated ( 5 min , 95°C ). After cooling on ice for 2 min , 700 μL chloroform:isoamyl alcohol (24:1) was added and the mixture incubated (10 min , room temperature) while shaking gently. The mixture was centrifuged (10 min , 4°C , maximal speed). The upper phase was transferred to a fresh tube and 1 volume chloroform:isoamyl alcohol was added. After repeating the centrifugation step, the upper phase was transferred and precipitated with 3 volumes of 99.5 % ethanol and 1/2 volume 7.5 M ammonium acetate and (time series only) 1 μL RNA-grade glycogen at −20°C over night. The RNA was pelleted by centrifugation (30 min , 4°C , maximum speed), washed twice with 70 % ethanol and resuspended in 30 μL RNase-free water. Volumes were adjusted to contain 2 μg of nucleic acid (Nanodrop), and DNA was removed by DNaseI (ThermoFisher: EN0525) according to the manufacturer's specifications but at 2x reaction buffer concentration. RNA was extracted as above but using 1/10 volume of 3 M sodium acetate (pH 5.3) instead of ammonium acetate for precipitation. The Illumina Ribo-Zero Plus rRNA Depletion Kit was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was evaluated with the RNA Pico 6000 kit on the Agilent 2100 Bioanalyzer. RNA was free of detectable rRNA. Preparation of cDNA libraries was performed according to the manufacturer's instructions for the TruSeq stranded mRNA kit (Illumina, San Diego, CA, United States). Subsequently,
ENA first public
2021-12-06
ENA last update
2021-12-06
External Id
SAMEA10146955
INSDC center alias
Center for Biotechnology, CeBiTec, University of Bielefeld, Universitatsstrasse 27, 33699 Bielefeld, Germany
INSDC center name
Center for Biotechnology, CeBiTec, University of Bielefeld, Universitatsstrasse 27, 33699 Bielefeld, Germany
INSDC first public
2021-12-06T00:11:47Z
INSDC last update
2021-12-06T00:11:47Z
INSDC status
public
Submitter Id
E-MTAB-10949:gyrA-kd_rep3
broker name
ArrayExpress
genotype
Crispr-mediated gyrA knockdown
organism part
whole organism
replicate
rep3
sample name
E-MTAB-10949:gyrA-kd_rep3
strain
gyrA-kd