2.5 Amplification
of Adapter-Ligated
Library
1. SureSelect
XT Reagent Kit, MSQ, 16 (Agilent).
2. Herculase II Fusion DNA Polymerase (Agilent).
3. Precapture PCR Mix (35μL per sample): 21μL of NucleaseFree Water, 1.25μL of SureSelect Primer 1.0 (forward), 1.25μL
of SureSelect Indexing Pre-Capture PCR Primer (reverse),
10μL of Herculase II 5Â reaction buffer, 0.5μL of dNTP
(100 mM), and 1μL of Herculase II polymerase.
4. Agencourt AMPure XP beads (Beckman Coulter).
5. DynaMag-96 Side magnet (Life Technologies).
6. 70% ethanol (see Note 2).
7. Nuclease-free water.
8. Thin-wall 8-strip tubes and caps (Thermo Scientific).
9. 2200 TapeStation Nucleic Acid System (Agilent).
10. D1000 ScreenTape and D1000 reagents (Agilent).
11. Thermal cycler, e.g., SureCycler 8800, with 96-well module.
12. 5–50μL 8-channel pipette and 20–200μL 8-channel pipette.
2.6 Hybridization
of Adapter-Ligated
Library to RNA Baits
1. SureSelect
XT Reagent Kit, MSQ, 16 (Agilent) (see Note 3).
2. SureSelect Block Mix (5.6μL per sample): 2.5μL of SureSelect
Indexing block #1 (green cap), 2.5μL of SureSelect block #2
(blue cap), and 0.6μL of SureSelect GA Indexing block #3
(brown cap).
3. Hybridization Buffer (20μL per sample): 12.5μL of SureSelect
Hyb #1 (orange cap), 0.5μL of SureSelect Hyb #2 (red cap),
5μL of SureSelect Hyb #3 (yellow cap), and 6.5μL of SureSelect Hyb #4 (black cap).
4. M. tuberculosis RNA capture baits (Oxford Gene Technologies)
(see Note 4).
5. Vacuum concentrator.
6. Thin-wall 96-well PCR Plates and caps (Thermo Scientific).
7. 5–50μL 8-channel pipette.
8. Thermal cycler, e.g., SureCycler 8800, with 96-well module
(Agilent).
9. 1.5 mL tube heat block set at 65 ˚C.
10. Dynabeads MyOne Streptavidin T1 (Life Technologies).
11. DynaMag-2 magnetic stand (Life Technologies).
12. Nutator (BD).
13. Nuclease-free water.
14. Vortex mixer.
15. Microfuge.
16. DNA LoBind 1.5 mL tubes (Eppendorf).
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Amanda Claire Brown
of Adapter-Ligated
Library
1. SureSelect
XT Reagent Kit, MSQ, 16 (Agilent).
2. Herculase II Fusion DNA Polymerase (Agilent).
3. Precapture PCR Mix (35μL per sample): 21μL of NucleaseFree Water, 1.25μL of SureSelect Primer 1.0 (forward), 1.25μL
of SureSelect Indexing Pre-Capture PCR Primer (reverse),
10μL of Herculase II 5Â reaction buffer, 0.5μL of dNTP
(100 mM), and 1μL of Herculase II polymerase.
4. Agencourt AMPure XP beads (Beckman Coulter).
5. DynaMag-96 Side magnet (Life Technologies).
6. 70% ethanol (see Note 2).
7. Nuclease-free water.
8. Thin-wall 8-strip tubes and caps (Thermo Scientific).
9. 2200 TapeStation Nucleic Acid System (Agilent).
10. D1000 ScreenTape and D1000 reagents (Agilent).
11. Thermal cycler, e.g., SureCycler 8800, with 96-well module.
12. 5–50μL 8-channel pipette and 20–200μL 8-channel pipette.
2.6 Hybridization
of Adapter-Ligated
Library to RNA Baits
1. SureSelect
XT Reagent Kit, MSQ, 16 (Agilent) (see Note 3).
2. SureSelect Block Mix (5.6μL per sample): 2.5μL of SureSelect
Indexing block #1 (green cap), 2.5μL of SureSelect block #2
(blue cap), and 0.6μL of SureSelect GA Indexing block #3
(brown cap).
3. Hybridization Buffer (20μL per sample): 12.5μL of SureSelect
Hyb #1 (orange cap), 0.5μL of SureSelect Hyb #2 (red cap),
5μL of SureSelect Hyb #3 (yellow cap), and 6.5μL of SureSelect Hyb #4 (black cap).
4. M. tuberculosis RNA capture baits (Oxford Gene Technologies)
(see Note 4).
5. Vacuum concentrator.
6. Thin-wall 96-well PCR Plates and caps (Thermo Scientific).
7. 5–50μL 8-channel pipette.
8. Thermal cycler, e.g., SureCycler 8800, with 96-well module
(Agilent).
9. 1.5 mL tube heat block set at 65 ˚C.
10. Dynabeads MyOne Streptavidin T1 (Life Technologies).
11. DynaMag-2 magnetic stand (Life Technologies).
12. Nutator (BD).
13. Nuclease-free water.
14. Vortex mixer.
15. Microfuge.
16. DNA LoBind 1.5 mL tubes (Eppendorf).
464
Amanda Claire Brown