10. Dry samples on thermocycler at 37
C for not more than 5 min
(see Note 10).
11. Add 32μL of nuclease-free water directly to each bead pellet.
Mix by vortexing and briefly spin tubes to collect contents.
Incubate for 2 min at room temperature.
12. Put tubes on magnetic rack, leave for 2–3 min to clear and
collect the supernatant (~30μL), and transfer to new 8-tube
strip.
13. Add 3μL of D1000 Sample Buffer and 1μL of D1000 ladder to
tube 1 to a new 8-tube strip (see Note 11).
14. Add 3μL of D1000 Sample Buffer and 1μL of sample to tubes
2–16 using 8-tube strips (see Note 12), add caps and vortex,
spin down for 1 min, and remove caps gently.
15. Load samples into TapeStation and run D1000 tape. Assess
quality and quantity; the peak height should be ~250–275 bp.
16. Samples can be stored overnight in the fridge at 4
C or at
À20
C for longer terms.
3.6 Hybridization
of Adapter-Ligated
Library to RNA Baits
(See Note 14)
1. Concentrate Precapture PCR samples using vacuum concentrator, as required, so they reach 147–220 ng/μL in nucleasefree water (see Note 15).
2. Using a 96-well plate (PLATE A), add 3.4μL (500-750 ng) of
each sample into separate wells of column 2 (see Note 16).
3. Prepare sufficient SureSelect Block Mix for all samples being
hybridized.
4. Add 5.6μL of SureSelect Block Mix to each sample well in
column 2 of the 96-well plate.
5. Add strip caps to column 2 and seal well; carefully check each
cap to ensure a complete seal.
6. Incubate plate(s) with samples in column 2 for 95 ˚C for 5 min
and then hold at 65 ˚C, using a thermocycler with a heated lid
(105 ˚C).
Table 1
Amplification of adapter-ligated library
Step
Temperature (
C)
Time
1. Initial denaturing
98
2 min
2. Denaturing
98
30 s
3. Annealing
65
30 s
4. Extension
72
1 min
5. Final extension
72
10 min
6. Cool down
4
Hold
Direct WGS of M. tb’
471
C for not more than 5 min
(see Note 10).
11. Add 32μL of nuclease-free water directly to each bead pellet.
Mix by vortexing and briefly spin tubes to collect contents.
Incubate for 2 min at room temperature.
12. Put tubes on magnetic rack, leave for 2–3 min to clear and
collect the supernatant (~30μL), and transfer to new 8-tube
strip.
13. Add 3μL of D1000 Sample Buffer and 1μL of D1000 ladder to
tube 1 to a new 8-tube strip (see Note 11).
14. Add 3μL of D1000 Sample Buffer and 1μL of sample to tubes
2–16 using 8-tube strips (see Note 12), add caps and vortex,
spin down for 1 min, and remove caps gently.
15. Load samples into TapeStation and run D1000 tape. Assess
quality and quantity; the peak height should be ~250–275 bp.
16. Samples can be stored overnight in the fridge at 4
C or at
À20
C for longer terms.
3.6 Hybridization
of Adapter-Ligated
Library to RNA Baits
(See Note 14)
1. Concentrate Precapture PCR samples using vacuum concentrator, as required, so they reach 147–220 ng/μL in nucleasefree water (see Note 15).
2. Using a 96-well plate (PLATE A), add 3.4μL (500-750 ng) of
each sample into separate wells of column 2 (see Note 16).
3. Prepare sufficient SureSelect Block Mix for all samples being
hybridized.
4. Add 5.6μL of SureSelect Block Mix to each sample well in
column 2 of the 96-well plate.
5. Add strip caps to column 2 and seal well; carefully check each
cap to ensure a complete seal.
6. Incubate plate(s) with samples in column 2 for 95 ˚C for 5 min
and then hold at 65 ˚C, using a thermocycler with a heated lid
(105 ˚C).
Table 1
Amplification of adapter-ligated library
Step
Temperature (
C)
Time
1. Initial denaturing
98
2 min
2. Denaturing
98
30 s
3. Annealing
65
30 s
4. Extension
72
1 min
5. Final extension
72
10 min
6. Cool down
4
Hold
Direct WGS of M. tb’
471