four quadrants within one of 24,384-well plates. Our 96 reverse
primers all incorporate 5
0 -overhangs containing the same Illumina P5 sequence, but are unique in a barcode indicating one
of the 96 wells within a 384-well plate quadrant. This scheme
provides 9216 unique combinations, putting an upper limit of
24,384-well plates (or 96 96-well plates) that can be pooled on
a single sequencing flow cell.
9. With fewer than 20 strains, this can be done using a 1 cm
cuvette spectrophotometer. If a spectrophotometer with
plate-reading capability is available and more strains are being
handled, it is convenient to pipette 100μL of each strain into
wells of a 96-well clear polystyrene plate and measure OD 600 ,
using a previously determined conversion factor to calculate
the equivalent 1 cm cuvette OD 600 .
10. We also add 2.6 fM of a second spike-in plasmid to the PCR
master mix, as a sentinel of performance of this step.
11. Each PCR reaction is 10μL, therefore roughly 1200 reaction
volumes (or about two 384-well plates with edge wells
excluded) can fit with SPRI beads in a 15 mL conical tube,
and 20,000 in a 50 mL conical tube. If processing a small pool
(less than one plate), 60 reactions will fit with SPRI beads in a
microcentrifuge tube.
References
1. Emmart EW (1945) The tuberculostatic action
of streptothricin and streptomycin with special
reference to the action of streptomycin on the
chorioallantoic membrane of the chick
embryo. Public Health Rep 60(48):1415
2. Bastian H (2006) Down and almost out in
Scotland: George Orwell, tuberculosis and getting streptomycin in 1948. J R Soc Med 99
(2):95–98
3. Andries K et al (2014) Acquired resistance of
Mycobacterium tuberculosis to Bedaquiline.
PLoS One 9(7):e102135
4. WHO (2018) Global Tuberculosis Report
2018. World Health Organization
5. Johnson EO et al (2019) Large-scale chemical
genetics yields new M. tuberculosis inhibitor
classes. Nature 571(7763):72–78
6. Johnson EO et al (2020) Large-Scale Chemical-Genetic Strategy Enables the Design of
Antimicrobial Combination Chemotherapy
in Mycobacteria. ACS Infect Dis 6(1):56–63
7. Schnappinger D, Ehrt S (2014) Regulated
expression systems for mycobacteria and their
applications. Microbiol Spectr 2(1):MGM20018-2013
8. Schnappinger D, O’Brien KM, Ehrt S (2015)
Construction of conditional knockdown
mutants in mycobacteria. In: Mycobacteria
Protocols. Humana Press, New York, NY, pp
151–175
9. Kim J-H et al (2013) A genetic strategy to
identify targets for the development of drugs
that prevent bacterial persistence. Proc Natl
Acad Sci U S A 110(47):19095–19100
10. Kim J-H et al (2011) Protein inactivation in
mycobacteria by controlled proteolysis and its
application to deplete the beta subunit of RNA
polymerase.
Nucleic
Acids
Res
39
(6):2210–2220
11. Risso D et al (2014) Normalization of
RNA-seq data using factor analysis of control
genes or samples. Nat Biotechnol 32
(9):896–902
12. Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome
Biol 11(10):R106
13. Leek JT et al (2010) Tackling the widespread
and critical impact of batch effects in highthroughput data. Nat Rev Genet 11
(10):733–739
Proteolytic Hypomorphs for Compound Mechanism of Action
341
primers all incorporate 5
0 -overhangs containing the same Illumina P5 sequence, but are unique in a barcode indicating one
of the 96 wells within a 384-well plate quadrant. This scheme
provides 9216 unique combinations, putting an upper limit of
24,384-well plates (or 96 96-well plates) that can be pooled on
a single sequencing flow cell.
9. With fewer than 20 strains, this can be done using a 1 cm
cuvette spectrophotometer. If a spectrophotometer with
plate-reading capability is available and more strains are being
handled, it is convenient to pipette 100μL of each strain into
wells of a 96-well clear polystyrene plate and measure OD 600 ,
using a previously determined conversion factor to calculate
the equivalent 1 cm cuvette OD 600 .
10. We also add 2.6 fM of a second spike-in plasmid to the PCR
master mix, as a sentinel of performance of this step.
11. Each PCR reaction is 10μL, therefore roughly 1200 reaction
volumes (or about two 384-well plates with edge wells
excluded) can fit with SPRI beads in a 15 mL conical tube,
and 20,000 in a 50 mL conical tube. If processing a small pool
(less than one plate), 60 reactions will fit with SPRI beads in a
microcentrifuge tube.
References
1. Emmart EW (1945) The tuberculostatic action
of streptothricin and streptomycin with special
reference to the action of streptomycin on the
chorioallantoic membrane of the chick
embryo. Public Health Rep 60(48):1415
2. Bastian H (2006) Down and almost out in
Scotland: George Orwell, tuberculosis and getting streptomycin in 1948. J R Soc Med 99
(2):95–98
3. Andries K et al (2014) Acquired resistance of
Mycobacterium tuberculosis to Bedaquiline.
PLoS One 9(7):e102135
4. WHO (2018) Global Tuberculosis Report
2018. World Health Organization
5. Johnson EO et al (2019) Large-scale chemical
genetics yields new M. tuberculosis inhibitor
classes. Nature 571(7763):72–78
6. Johnson EO et al (2020) Large-Scale Chemical-Genetic Strategy Enables the Design of
Antimicrobial Combination Chemotherapy
in Mycobacteria. ACS Infect Dis 6(1):56–63
7. Schnappinger D, Ehrt S (2014) Regulated
expression systems for mycobacteria and their
applications. Microbiol Spectr 2(1):MGM20018-2013
8. Schnappinger D, O’Brien KM, Ehrt S (2015)
Construction of conditional knockdown
mutants in mycobacteria. In: Mycobacteria
Protocols. Humana Press, New York, NY, pp
151–175
9. Kim J-H et al (2013) A genetic strategy to
identify targets for the development of drugs
that prevent bacterial persistence. Proc Natl
Acad Sci U S A 110(47):19095–19100
10. Kim J-H et al (2011) Protein inactivation in
mycobacteria by controlled proteolysis and its
application to deplete the beta subunit of RNA
polymerase.
Nucleic
Acids
Res
39
(6):2210–2220
11. Risso D et al (2014) Normalization of
RNA-seq data using factor analysis of control
genes or samples. Nat Biotechnol 32
(9):896–902
12. Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome
Biol 11(10):R106
13. Leek JT et al (2010) Tackling the widespread
and critical impact of batch effects in highthroughput data. Nat Rev Genet 11
(10):733–739
Proteolytic Hypomorphs for Compound Mechanism of Action
341