description: Protocols: The strain used in this study is the original clinical motile isolate of NCTC 11168 from the National Collection of Type Cultures. The strain was routinely cultured on Campylobacter blood-free selective agar supplemented with CCDA (containing Cefoperazone and Amphotericin B) (Oxoid). After 24 hours of incubation, a loop of NCTC 11168 culture was transferred onto and incubated overnight on agar plates containing cation-adjusted Mueller-Hinton 2 (MH2) (Sigma-Aldrich) 1.5% agar (BactoTMAgar). %(kindly donated by Professor Charles Penn from the University of Birmingham)The culture was incubated at 37 \textdegree{}C in a variable atmosphere incubator (VAIN) (Whitley VA500 workstation cabinet), with the following atmospheric content: 90 % N2 (v/v), 6 % CO2 (v/v), 4 % O2 (v/v)). 3.5 mL of cell culture was killed by adding 0.5 volumes of cold (4 oC) killing buffer (20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 20 mM NaN3). Cell cultures were spun down at 5000-10000 x g for 3-5 minutes, then snap frozen in liquid nitrogen and stored at -80 oC until further use. Sample pellets were placed on ice before extraction. Workspace and pipettes were cleaned with RNaseZapTM (Sigma-Aldrich) prior to RNA extraction. Nuclease-free molecular biology grade water (Sigma-Aldrich), RNase-free filter tips, RNase-free eppendorf tubes (Sarstedt), and RNase-free 15 mL or 50 mL tubes (Falcon) were used throughout RNA extraction protocols. All centrifugation steps were carried out on Sigma 1-14K microcentrifuge (Sigma).The pellet was resuspended in 600 ul of LETS buffer (0.1M LiCl, 0.01M Na2EDTA, 0.01M Tris-Cl (pH 7.4), 0.2%SDS) and transferred to Lysing Matrix B, 0.1 mm tubes (MP biomedicalsTM). The tubes were placed in a FastPrep Homogenizer (MP biomedicalsTM) to perform 3 rounds of bead-beating at a speed of 6.0 m/s for 40 seconds, with a 5 minutes pause between rounds. The samples were then centrifugated at 13000 x g for 10 minutes at 4 oC. Afterwards, proteins and nucleic acids were separated from the cell debris by centrifuging at 13000 rpm at 4 oC for 10 minutes. RNA was phase-separated by 2 rounds of 125:24:1 Phenol/Chloroform/isoamylalcohol (PCI) and one round of 24:1 chloroform/isoamyl alcohol. The upper phase was mixed with 0.1 volume of 3M sodium acetate (pH 5.2) and 1 volume of isopropanol for overnight precipitation at -20 oC.After overnight precipitation, samples were centrifugated at top speed at 4 oC for 15 minutes. The supernatant was removed and pellets washed (without resuspending) with fresh 70% ethanol twice. Pellets were air-dried for 15 minutes before resuspending in 50 µL of RNase-free water. The resuspended RNA was incubated on ice for three hours, then 30 minutes at room temperature. This protocol was adapted from Shishkin et al. 2015.
attributes.tag: ENA first public
attributes.value: 2022-05-27
attributes.tag: ENA last update
attributes.value: 2022-05-27
attributes.tag: External Id
attributes.value: SAMEA12280484
attributes.tag: INSDC center alias
attributes.value: UNIVERSITY OF WARWICK
attributes.tag: INSDC center name
attributes.value: UNIVERSITY OF WARWICK
attributes.tag: INSDC first public
attributes.value: 2022-05-27T20:14:43Z
attributes.tag: INSDC last update
attributes.value: 2022-05-27T20:14:43Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11309:black_cold_4_S4
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: genotype
attributes.value: wild type
attributes.tag: growth condition
attributes.value: cold
attributes.tag: sample name
attributes.value: E-MTAB-11309:black_cold_4_S4
attributes.tag: sub_species
attributes.value: jejuni