Figure 1. Schematic representation of the non-typeable Haemophilus influenzae tn-seq mutant library screen for survival and growth in environmental air

Materials and Reagents

1. 1 U/μl Calf Intestinal Alkaline Phoshatase (CIAP) (New England Biolabs, catalog number: M0290S )
   
2. Chloroform: isoamyl alcohol
   
3. Phenol: chloroform: isoamayl alcohol
   
4. Milli-Q water
   
5. 10 mM dNTP mix (New England Biolabs, catalog number: N0447S )
   
6. 1 mM dNTP mix
   
7. Absolute ethanol
   
8. 70 % Ethanol
   
9. 10 mg/ml Glycogen
   
10. 2 U/μl MmeI restriction enzyme (New England Biolabs, catalog number: R0637S )
    
11. 10x NEBuffer 4
    
12. 32 mM S-denosylmethionine
    
13. 3 M NaAc (pH 5.3)
    
14. 5 M NaCl
    
15. 2 U/μl Phusion DNA polymerase (New England Biolabs, catalog number: M0530S )
    
16. 5x Phusion HF buffer
    
17. 10 U/μl T4 DNA ligase (New England Biolabs, catalog number: M0202S )
    
18. 20 U/μl T4 DNA ligase
    
19. 10x T4 DNA ligase buffer
    
20. 2.5 U/μl T4 DNA polymerase (New England Biolabs, catalog number: M0203S )
    
21. 10x T4 DNA polymerase buffer
    
22. T4 polynucleotide kinase (3' phosphatase minus) (New England Biolabs, catalog number: M0236S )
    
23. 100x TE buffer
    
24. 1 M NaOH
    
25. 50 % Glycerol
    
26. 1 mM DTT
    
27. 5 M NaCl
    
28. 1 M MgCl₂
    
29. 10 mg/ml BSA
    
30. 5 U/ml E.coli DNA ligase (New England Biolabs, catalog number: M0205S )
    
31. 10x E.coli DNA ligase buffer
    
32. 1 M Hepes (pH 7.9)
    
33. HimarC9 transposase
    
34. M-IV medium (Herriott et al., 1970)
    
35. 1 mg/ml Hemin (Sigma-Aldrich, catalog number: H9039 )
    
36. 10 mg/ml Nicotinamide adenine dinucleotide (NAD) (Sigma-Aldrich, catalog number: N7004 )
    
37. Brain heart infusion medium (BHI) (BD Biosciences, catalog number: 237500 )
    
38. Supplemented BHI, BHI medium containing 10 μg/ml Hemin and 2 μg/ml NAD
    
39. Bacto-agar (BD Biosciences, catalog number: 212030 )
    
40. Supplemented BHI plates, sBHI containing 1.5% bacto agar
    
41. PBS
    
42. 100 mg/ml RNase A (Roche Diagnostics, catalog number: 10109142001 )
    
43. Qiagen Genomic-tip 20/G columns (QIAGEN, catalog number: 10223 )
    
44. Qiagen Genomic DNA buffer set (QIAGEN, catalog number: 19060 )
    
45. Minelute Reaction Cleanup Kit (QIAGEN, catalog number: 28204 )
    
46. Qubit dsDNA BR assay (Life technologies, catalog number: Q32850 )
    
47. Acceptor DNA
    Any type of DNA can serve as acceptor for in vitro mariner transposition. The most common types of acceptor DNA are: Chromosomal DNA of the target strain (High quality DNA is required, preferably isolated with Qiagen Genomic Tip columns) or PCR products of target genes or regions.
    
48. Donor DNA
    Any type of DNA that carries a mariner transposon with MmeI restriction site in the inverted repeat can serve as donor for transposon in the in vitro mariner transposition reaction. Used pGSF8 plasmid, carrying transposon with spectinomycin resistance cassette, suitable for GAF and TnSeq (Langereis et al., 2013).
    
49. Primers used for sequence adapters ligation and PCR amplification (see Appendixes)
    

Equipment

1. Pipet tips: 0.5-10 μl, 2-20 μl, 20-200 μl 100-1000 μl
2. 15 cm dishes
   
3. Heating block for incubations ranging from 16 °C and 75 °C (Grant QBD2)
   
4. Microcentrifuge for 1.5 ml tubes (Eppendorf, model: 5417R )
   
5. Centrifuge for 50 ml tubes (Eppendorf, model: 5810 )
   
6. T100 thermal cycler (Bio-Rad Laboratories)
   
7. Nanodrop spectrophotometer (Thermo Fisher Scientific, Nanodrop, model: ND1000 )
   
8. Incubator with 5% CO₂ (BINDER GmbH, model: CB 150 )
   
9. Qubit Fluorometer (Life Technologies)
   
10. Bioanalyser (Agilent Technologies)
    

Procedure