Chapter 2. Methodological section
1. Methods used to study M. extorquens model strains
During this thesis, eight cDNA banks were constructed using RNA extracted from M. extorquens CM4, the model strain for growth with chloromethane (2 biological independent replicates), and from M. extorquens DM4, the model strain for growth with dichloromethane (2 biological independent replicates). In addition, RNAs were extracted from both strains grown using a methylotrophic reference substrate, methanol (2 biological independent replicates per strain).
1.1. Aerobic methylotrophic growth
Cultured of 220 mL, were performed into M3 medium defined for Methylobacterium with 10 mM of carbon (Muller et al., 2011a). To prepare 1 liter of M3 medium, 1.9 g of KH2PO4, 6.39 g of Na2HPO4·2 H2O, 0.1 g of MgSO4·7 H2O and 0.2 g de (NH4)2SO4 are dissolved into 800 mL of milliQ water. After autoclaving, 1 mL of Ca(NO3)2·4 H2O at 25 g.L-1 is added with 1 mL of a trace solution. This solution contains H2SO4 concentrated (95 %) at 9.2 g.L-1, Fe(II)SO4·7 H2O at 1.0 g.L-1, MnSO4·H2O at 1.0 g.L-1, Na2MoO4·H2O at.25 g.L-1, H3BO3 at 0,1 g.L
-1, CuCl2·2 H2O at 0.28 g.L-1, ZnSO4·7 H2O at 0.53 g.L-1, NH4VO3 at 0.1 g.L-1, Co(NO3)2·6 H2O à 0.25 g.L-1, NiSO4·7 H2O at 0.1 g.L-1. Growth conditions and total RNA extractions are described in the article “Contribution of the Core and Variable Genome to the Transcriptomes of two M extorquens Strains Grown with Chloromethane, Dichloromethane or Methanol” (see Chapter 3, section 3.2.).
1.2. RNA preparation
The total RNA extraction protocol is described in the Chapter 3 in the Materials and Methods section. No kit was utilized to the RNAs extraction, but a protocol with a phenol/chloroform step. These methods have some advantages in comparison with kits; no restriction in the total amount of RNA extracted, and no size selection. Once total RNA has been extracted, 2 additional steps before cDNA bank construction are needed: rRNA removal and RNA quality check.
1.2.1. DNA removal
A DNase treatment was used to remove DNA contaminants (Turbo DNAse Ambion Thermo Fischer Scientific). A total of 3 µL of Turbo DNAse was added (2U/µL) with 2 µL of RNAsin (Promega), 10 µL of TURBO™ buffer (10 X; Ambion) and 78µL of milli DEPC-treated water.
The reactionnal mix is incubated two times, at 30 minutes at 37°C, with 2µL of TURBO™
DNase added between those two incubations. Evaluation of the efficiency of DNA-free RNA extracts was checked by PCR with three sets of primers targeting the 16S RNA-encoding gene rrsA and cliA present in both strains. Additional PCR targeted genes cmuA and dcmA, which are specific of strains CM4 and DM4 respectively, were also used (see article
“Contribution of the Core and Variable Genome to the Transcriptomes of two M. extorquens Strains Grown with Chloromethane, Dichloromethane or Methanol” (see Chapter 3, section 3.2.). PCR conditions are summarized in Table 2.1.
Table 2.1. PCR amplification program to validate DNA removal
Mix Volume
(µL) PCR program
H2O milliQ water 17.6 Pre-denaturation, 2 min at 95°C
PCR buffer (Biolabs, 10X) 2.5
40 cycles
Denaturation, 30 sec at 94°C
dNTP 20mM each 0.25 Annealing, 45 sec at 60°C
Forward primer (20µM) 1 Extension, 30 sec at 72°C
Reverse primer (20µM) 1 Final extension, 10 min at 72°C
Taq polymerase 0.1
DNA 1
1.2.2. rRNA removal
The abundance of ribosomal RNA (rRNA) accounts for 95-98 % of total RNA in bacteria. Thus, a step to efficiently remove rRNA is necessary to achieve optimal coverage of mRNA, good detection sensitivity and reliable results. The necessity to optimize efficient rRNA removal methods for RNA sequencing in other GC-rich bacteria has been described previously (Peano et al., 2013). Two types of rRNA depletion approaches exist: the first one is based on a selective degradation of rRNAs by an enzyme recognizing specifically the 5’ monophosphate extremity of rRNAs, whereas mRNAs are protected by their triphosphates extremities (Evguenieva-Hackenberg and Klug, 2011). The second method relies on subtractive hybridization with probes specifically designed to target rRNA conserved regions (van Dijk et al., 2014). This second method was the most efficient in rRNA depletion of samples extracted from M. extorquens strains as tested at the Génoscope in Evry (experiments
107 performed by Béatrice Ségurens and Adriana Albertini). Extracted RNA samples were treated with the RiBoZero Magnetic kit Gram-negative bacteria (Tebu-Bio). Evaluation of the efficiency of RNA removal was checked by PCR with primers targeting the 16S RNA-encoding gene rrsA and cliA as a control. Despite the validation of rRNA removal by PCR and bioanalyzer profil, 14 up to 34 % of the RNAseq reads mapped to rRNA (Chapter 3 (section 3.2, Table 1). Thus, the RiboZero Magnetic Kit Gram-negative bacteria is not optimized to target M. extorquens rRNA (data confirmed by the Tebu-Bio company).
1.2.3. RNA quality control
Enzymes that degrade RNA, ribonucleases (RNases), are so ubiquitous (reagents, skin, dust) that working in an RNase-free environment can be challenging. The integrity of the extracted RNA was systematically checked using the Agilent biotechnology 2100 Bioanalyzer System.
This system is based on classical gel electrophoresis adapted to a chip format. The RNA nano and RNA pico kits are used to detect RNA in the range of 5-500 ng.µL-1 and 50-5000 pg.µL-1 respectively. Each RNA sample is mixed to a fluorescent dye and loaded into a well connected to micro-channels for capillary electrophoresis. The dye binds to nucleic acids, then RNA-dye complex are detected by laser-induced fluorescence. Fluorescence is recorded for nucleic acid visualization. RNA fragments are separated based on their size and fragment size assignment is deduced from the electrophoregram of a co-migrating RNA size ladder (Figure 2.1).
Figure 2.1. Electrophoregram of RNAs extracted from M. extorquens CM4 growing with chloromethane
Peak area is assigned in arbitrary fluorescence units (FU). (A) RNA nano kit-based electrophoregram of RNA before rRNA depletion (RIN value of 6.7). In M. extorquens an additional band is observed. (B) RNA pico kit-based electrophoregram of RNA after the rRNA depletion (RIN value of 2.6).
Profiles generated on the Agilent 2100 Bioanalyzer System yield information on concentration, allow a visual inspection of RNA integrity, and generate ribosomal ratios (16S versus 23S rRNA). Two kits are available for RNA analysis: RNA nano and RNA pico kits, depending on their sensibility (5-500 ng.µL-1 and 50-5000 pg.µL-10 respectively). The manufacturer Agilent Technologies has introduced the RNA Integrity Number (RIN). The RIN is an algorithm for assigning integrity values to RNA measurements: a value of 1 means that RNAs are totally degraded while a value of 10 means “perfect” RNAs integrity with no detected degradation. The RIN value is defined by a complex calculations fixed by the software using the ratio 16S/23S. Moreover, the RIN value should be use with caution. In some bacteria, RNAs can be naturally fragmented, as described in M. extorquens (Zahn et al., 2000; Evguenieva-Hackenberg, 2005). This maturation will lead to the 23S fractionation, illustrating by the apparition a new peak between those for the 16S and 23S, as observed in Figure 2.1.
1.3. Construction of the directional cDNA bank
To construct the cDNA banks, Béatrice Segurens and Adriana Albertini performed preliminary tests at the Génoscope in Evry. Three protocols were tested basing on the TruSeq Small RNA, the TruSeq stranded mRNA, and a Génoscope “house” protocol. The TruSeq stranded mRNA LT kit (Illumina) was chosen. The directional bank relies on dUTP incorporation during the second strand synthesis. This will avoid the amplification of the second strand, keeping only information from the first strand cDNA. The addition of two different adapters, in 3’ and 5’ extremities will keep the strand orientation. Fifty to sixty ng of rRNA-depleted RNA were used to construct directional cDNA banks as described in Figure 2.2.
109 Figure 2.2. Directional cDNA bank construction
The TruSeq stranded mRNA kit (Illumina) was used. (1) RNAs were primed with random hexamers to synthetize the first cDNA strand by reverse transcription. (2) The complementary cDNA strand was synthetized with the incorporation of dUTP instead of dTTP, whereas the RNA template was degraded; (3) A single adenosine nucleotide (A) was added to the 3’ ends of the blunt extremity of each cDNA fragment to prevent them from ligation with other cDNA fragments; (4) Ligation of multiple indexing adapters to both the 5’
and 3’ ends of the cDNA fragments; (5) Selectively enriched DNA fragments: PCR amplification and treatment with magnetic beads (AMP Xpure, Agencourt, Agilent) for PCR product size selection; (6) cDNAs with a size around 260 bp were hybridized to surface-bound primers in the flow cell. Then sequencing can start.
The cDNA bank constructions were validated if the final product had an expected size around 260 bp using the Agilent DNA 1000 kit. An example of an electrophoregram of cDNA with a DNA chip is shown (Figure 2.3). Validated cDNA banks were sent to the Génoscope to be sequenced in paired-end with the HiSeq2000.
Figure 2.3. Validation of cDNA bank constructions with the 2100 Bioanalyzer