SALVACIÓN UNIVERSAL
LA IGLESIA VERDADERA OFRECE LA SALVACIÓN A TODOS
Transcriptomic data for prophage BTP1 (Chapter 5.2) was taken from the complete D23580 transcriptome dataset including identification of transcription start sites (TSS) using differential RNA-seq (Canals et al., 2018; Hammarlöf et al., 2017), where experiment design was based on that used in Kröger et al. (Kröger et al., 2012, 2013).. A description of the experimental conditions associated with the data presented in this thesis is given in Table 2.8. For analysis of prophage-regulated genes (Chapter 5.3), only the conditions ESP, inSPI2 and anaerobic growth were used.
2.9.2 RNA extraction
The protocol is essentially as described (Kröger et al., 2013). Four or 5 OD600 units were removed from bacterial cultures, and cellular transcription was stopped using 0.4X culture volume of a 5% phenol (pH 4.3) 95% ethanol “stop” solution (Sigma P4557 and E7023, respectively). Cells were stabilised on ice in stop solution for at least 30 minutes before cells were harvested at 7,000 x g for 10 minutes at 4°C. At this point pellets were either stored at -80°C, or RNA was immediately extracted. To isolate RNA, pellets were resuspended in 1 ml of TRIzolTM Reagent (Invitrogen). 400 µL of chloroform was added and the samples were immediately and thoroughly mixed by inversion. Samples were moved to a Phase-lock tube (5 Prime) and the aqueous and organic phases were separated by centrifugation at 13,000 rpm for 15 minutes at room temperature in a table top centrifuge. The aqueous phase was moved to a new 1.5 ml tube and the RNA was precipitated using isopropanol for 30 minutes at room temperature followed by centrifugation at 21,000 x g for 30 minutes at room temperature. The RNA pellet was rinsed with 70% ethanol followed by centrifugation at 21,000 x g rpm for 10 minutes at room temperature. The RNA pellet was air-dried for 15 minutes and resuspended in DEPC-treated water at 65°C with shaking at 900 rpm on a Thriller thermoshaker (Peqlab) for 5 minutes with occasional vortexing. RNA was kept on ice whenever possible and RNA was stored at -80°C. RNA concentration was measured using a nanodrop ND-1000 Spectrophotometer and RNA quality was inspected visually using a 2100 Bioanalyser (Agilent).
2.9.3 cDNA library preparation and RNA-seq
Strand-specific cDNA library preparation and high throughput cDNA sequencing (RNA-seq) of wild-type D23580 and isogenic prophage mutants was performed on DNase I digested total RNA by Vertis Biotechnologie AG (Freising, Germany). Strains and conditions in which they were grown before RNA extraction are detailed in Table 5.1. RNA was not depleted for ribosomal RNA. RNA was fragmented by sonication
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and poly(A)-tails were added to each fragment by poly(A) polymerase. The 5’ end of each fragment was de-phosphorylated using tobacco acid pyrophosphatase (TAP). An RNA adaptor, containing a 6-10 nucleotide bar-code was ligated to the 5’ end of each fragment to allow for identification of each RNA fragment after sequencing. First strand cDNA synthesis was performed using oligo(dT) priming and Moloney murine leukaemia virus reverse transcriptase (M-MLV RT). The resulting cDNA was amplified by PCR to approximately 20-30 ng/µL using a high fidelity DNA polymerase. cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analysed by capillary electrophoresis.
cDNA prepared by Vertis Biotechnologie AG was sequenced on an Illumina Nextseq 500 platform using 75 bp read lenghts. The strains and conditions under which they were grown before RNA was extracted are detailed in Table 5.1.
2.9.4 Generating Digoxigenin-labelled riboprobes
Digoxigenin (Dig)-labelled riboprobes were generated by in vitro transcription using T7 RNA polymerase using a Dig Northern Starter kit (Roche). A linear DNA template incorporating the antisense sequence to the transcript of interest and a T7 promoter sequence was first generated by PCR using primers shown in Table 2.4 and a high fidelity enzyme as previously described. The DNA template was purified by gel extraction according to the manufacturer’s instructions and 100-200 ng of DNA template was used in each in vitro transcription reaction. Linear DNA template was combined with 1X labelling mix, which contains unlabelled nucleotides and DIG-11- UTP, 1X transcription buffer and 40 units of T7 polymerase in a final volume of 20 µL. Labelling transcription mixes were incubated at 42°C for 1 hour. 20 U of DNase I was added to remove template DNA and the reactions were incubated at 37°C for 15 minutes. The reaction was stopped by addition of 400 mM EDTA (pH 8.0) and labelled riboprobes were stored at -20°C.
2.9.5 Northern Blotting
Total RNA after extraction as described in 2.9.2 was separated based on its size by electrophoresis through an 8.3 M Urea, 1X TBE (Tris Borate EDTA) 7% polyacrylamide gel and a denaturing 20 mM guanidine thiocyanate 1.5% agarose gel. Generally 1-10 µg of RNA was mixed with an equal volume of 2X Urea-Blue denaturing buffer (0.025% xylene cyanol, 0.025% bromophenol blue and 50% urea) and samples were heat-denatured at 90°C for 5 minutes and chilled on ice before loading. 4 µl of Low Range ssRNA ladder (NEB) or 5 µl RNA molecular weight marker I DIG-labeled (Roche) were treated in the same way as the RNA samples in order to
63 allow approximation of detected transcript length. Samples were run in 1X TBE running buffer at a constant voltage of 120V for denaturing polyacrylamide gels or 80 V (at 4°C) for denaturing agarose gels.
Separated RNA was transferred from polyacrylamide gels to positively charged nylon membranes (Roche, cat. 11 209 272 001) using the Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (BioRad) at a constant amplitude of 125 mA for 30 minutes at 4°C. For denaturing agarose gels, separated RNA was transferred to positively charged nylon membranes using overnight capillary transfer in 20X saline- sodium citrate (SSC) buffer as described in the DIG Application Manual (Roche). RNA was UV-crosslinked to the membranes in a CL-1000 UV-crosslinker (UVP) set to 3600 (360,000 µJ/cm2). The membrane was equilibrated in hybridisation buffer for 1 hour at 68°C in pre-warmed DIG Easy Hyb solution (Roche) in a rotating hybridisation oven. 5 µl (approximately 1.25 µg) of riboprobe was heat denatured in 5 ml of DIG Easy Hyb solution at 68°C for 30 minutes and added to the membrane for hybridisation overnight at 68°C in the rotating hybridisation oven. The membrane was washed twice for a total of 10 minutes in low stringency wash buffer 1 (2X SSC buffer, 0.1% SDS) at room temperature with rocking on a see-saw rocker (Stuart) at room temperature, followed by 2 washes for a total of 30 minutes in high stringency wash buffer 2 (0.1X SSC buffer, 0.1% SDS) with rocking at 68°C. Non-specific sites on the membrane were blocked using 1X blocking buffer (casein-based blocking buffer supplied by Roche and diluted 10-fold in maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl adjusted to pH 7.5 using NaOH pellets)) for 30 minutes at room temperature with rocking. Alkaline phosphatase conjugated polyclonal anti-digoxigenin Fab-fragment was diluted 1:10,000 in 1X blocking buffer and immunological detection of the membrane proceeded for 30 minutes at room temperature with rocking. The membrane was then washed twice for a total of 30 minutes in wash buffer (maleic acid buffer, 0.3% Tween-20). The membrane was incubated for 5 minutes in detection buffer (0.1M Tris-HCl, 0.1M NaCl, pH 9.5) and CDP-starTM (Tropix) was used as the chemiluminescent substrate. Enzymatic de-phosphorylation of CDP-star by alkaline phosphatase results in light emission which was visualised using an ImageQuant Las 4000 Imager.