Illumina MiSeq sequencing; Cappable-seq data for discovering condition-specific primary transcriptome in Campylobacter jejuni
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 L of RNase-free water. The resuspended RNA was incubated on ice for three hours, then 30 minutes at room temperature.  RNA samples from all three biological replicates from each condition were diluted to around 100 ng/ul and pooled together for Cappable-seq processing detailed in Ettwiller et al. A control sample was used which omitted the Streptavidin capture step.The libraries were created from the Cappable-seq processed RNA pool using NEBNext Small RNA Library Prep Set for Illumina (NEB). Note that the control pool and the Cappable-seq enriched pool were PCR amplified for 12 and 18 cycles respectively.