2.6 Quality Control
1. Molecular biology grade water.
2. Capillary electrophoresis analyzer (e.g., Agilent Bioanalyzer or
TapeStation) and associated high-sensitivity DNA chip.
2.7 Barcode
Counting
1. NGS platform (e.g., Illumina MiSeq or HiSeq 2500) and
associated reagent kits.
2. FASTX Toolkit (http://hannonlab.cshl.edu/fastx_toolkit),
HTSeq [14], or Bartender [15].
2.8 Fitness Inference
1. R.
2. R
package ConcensusGLM
(https://github.com/bro
adinstitute/concensusGLM).
2.9 Mechanism
of Action
Interpretation
1. R.
2. R package glmnet [16].
3 Methods
3.1 Strain
Propagation
1. Thaw freezer stocks of strains.
2. Inoculate one aerosol-tight culture vessel containing 10 mL
M7H9 supplemented with selection agents with 100μL stock
and 1μg/mL ATC for each strain.
3. Incubate at 37
C with shaking at 200 rpm for 4–7 days, until
OD 600 ¼ 0.3–1.0.
3.2 Inoculation
and Incubation
1. Measure OD 600 of each strain (see Note 9).
2. Calculate the total culture volume required for the experiment.
Each well requires 40μL of pooled culture, therefore each
384-well plate requires 15.36 mL plus dead volume for the
reservoir. We usually make 1.5–2Â the minimum amount of
culture.
3. Calculate the culture volume of each strain required to give an
equal number of cells and a final OD 600 of 0.0025.
4. Add each culture to a conical tube and mix with a pipette.
5. Harvest cells by centrifugation and spin down at 3000 Â g for
10 min.
6. Pipette off as much supernatant as possible while being careful
not to disturb the cell pellet.
7. Resuspend the cell pellet in 40 mL sterile M7H9.
8. Repeat this wash.
9. Resuspend in 40 mL M7H9. Gently pipette mix the
suspension.
334
Eachan O. Johnson and Deborah T. Hung
1. Molecular biology grade water.
2. Capillary electrophoresis analyzer (e.g., Agilent Bioanalyzer or
TapeStation) and associated high-sensitivity DNA chip.
2.7 Barcode
Counting
1. NGS platform (e.g., Illumina MiSeq or HiSeq 2500) and
associated reagent kits.
2. FASTX Toolkit (http://hannonlab.cshl.edu/fastx_toolkit),
HTSeq [14], or Bartender [15].
2.8 Fitness Inference
1. R.
2. R
package ConcensusGLM
(https://github.com/bro
adinstitute/concensusGLM).
2.9 Mechanism
of Action
Interpretation
1. R.
2. R package glmnet [16].
3 Methods
3.1 Strain
Propagation
1. Thaw freezer stocks of strains.
2. Inoculate one aerosol-tight culture vessel containing 10 mL
M7H9 supplemented with selection agents with 100μL stock
and 1μg/mL ATC for each strain.
3. Incubate at 37
C with shaking at 200 rpm for 4–7 days, until
OD 600 ¼ 0.3–1.0.
3.2 Inoculation
and Incubation
1. Measure OD 600 of each strain (see Note 9).
2. Calculate the total culture volume required for the experiment.
Each well requires 40μL of pooled culture, therefore each
384-well plate requires 15.36 mL plus dead volume for the
reservoir. We usually make 1.5–2Â the minimum amount of
culture.
3. Calculate the culture volume of each strain required to give an
equal number of cells and a final OD 600 of 0.0025.
4. Add each culture to a conical tube and mix with a pipette.
5. Harvest cells by centrifugation and spin down at 3000 Â g for
10 min.
6. Pipette off as much supernatant as possible while being careful
not to disturb the cell pellet.
7. Resuspend the cell pellet in 40 mL sterile M7H9.
8. Repeat this wash.
9. Resuspend in 40 mL M7H9. Gently pipette mix the
suspension.
334
Eachan O. Johnson and Deborah T. Hung