3.5 Shearing DNA
1. Transfer 50 μl of extracted DNA into a 1.5 ml
microcentrifuge tube.
2. Place the microcentrifuge tubes in the rack of the cup horn’s
sonicator. Add ice-cold water into the cup horn.
3. Run the sonicator at 80% intensity with cycles of 10 s on pulse
and 5 s off pulse for a total of 3 min (see Note 12).
4. Check the size of the DNA fragments by running 2.5 μl of
sheared DNA on a 2% agarose gel (see Note 13).
3.6 Addition
of the C-Tail
1. Determine the DNA concentration using a spectrophotometer, and calculate the volume corresponding to 500 ng of DNA
(see Note 14).
2. Set up the reaction as described in Table 3 to add C-tails.
3. Run the following program on the thermal cycler: 1 h at 37
C
then 20 min at 75
C.
4. Clean samples with a mini-PCR purification kit, and elute the
DNA with 12 μl of buffer.
3.7 Nested PCR
1. Prepare PCR mixes for the first PCR round (Table 4).
2. Run the following program on the thermal cycler: 95
C for
2 min; 24 cycles: 95
C for 30 s, 60
C for 30 s, 72
C for
2 min; 72
C for 2 min.
3. Set up the reaction for the second round of PCR (Table 5). An
indexing primer with a different barcode sequence should be
used for each replicate.
4. Run the following program on the thermal cycler: 95
C for
2 min; 18 cycles: 95
C for 30 s, 60
C for 30 s, 72
C for
2 min; 72
C for 2 min.
5. Confirm the size of DNA fragments by running 3 μl of sheared
DNA on a 2% agarose gel (see Note 15).
6. Purify the PCR products with a PCR purification kit following
the manufacturer’s instructions. Elute the DNA with 30 μl
elution buffer.
3.8 Preparation
of the Genomic
Libraries
for Sequencing
1. Measure the DNA concentration using a fluorometer.
2. Calculate the volume of each library corresponding to 15 or
20 ng, and pool all libraries together (see Note 16).
3. Sequence genomic libraries as 64 bp single-end reads with the
standard commercial sequencing primer (IP seq) and the custom primer pMargent3 (see Note 17). Run “sequencing-bysynthesis” reactions on a next-generation sequencing platform.
102
Kristel Lourdault and James Matsunaga
1. Transfer 50 μl of extracted DNA into a 1.5 ml
microcentrifuge tube.
2. Place the microcentrifuge tubes in the rack of the cup horn’s
sonicator. Add ice-cold water into the cup horn.
3. Run the sonicator at 80% intensity with cycles of 10 s on pulse
and 5 s off pulse for a total of 3 min (see Note 12).
4. Check the size of the DNA fragments by running 2.5 μl of
sheared DNA on a 2% agarose gel (see Note 13).
3.6 Addition
of the C-Tail
1. Determine the DNA concentration using a spectrophotometer, and calculate the volume corresponding to 500 ng of DNA
(see Note 14).
2. Set up the reaction as described in Table 3 to add C-tails.
3. Run the following program on the thermal cycler: 1 h at 37
C
then 20 min at 75
C.
4. Clean samples with a mini-PCR purification kit, and elute the
DNA with 12 μl of buffer.
3.7 Nested PCR
1. Prepare PCR mixes for the first PCR round (Table 4).
2. Run the following program on the thermal cycler: 95
C for
2 min; 24 cycles: 95
C for 30 s, 60
C for 30 s, 72
C for
2 min; 72
C for 2 min.
3. Set up the reaction for the second round of PCR (Table 5). An
indexing primer with a different barcode sequence should be
used for each replicate.
4. Run the following program on the thermal cycler: 95
C for
2 min; 18 cycles: 95
C for 30 s, 60
C for 30 s, 72
C for
2 min; 72
C for 2 min.
5. Confirm the size of DNA fragments by running 3 μl of sheared
DNA on a 2% agarose gel (see Note 15).
6. Purify the PCR products with a PCR purification kit following
the manufacturer’s instructions. Elute the DNA with 30 μl
elution buffer.
3.8 Preparation
of the Genomic
Libraries
for Sequencing
1. Measure the DNA concentration using a fluorometer.
2. Calculate the volume of each library corresponding to 15 or
20 ng, and pool all libraries together (see Note 16).
3. Sequence genomic libraries as 64 bp single-end reads with the
standard commercial sequencing primer (IP seq) and the custom primer pMargent3 (see Note 17). Run “sequencing-bysynthesis” reactions on a next-generation sequencing platform.
102
Kristel Lourdault and James Matsunaga