Genomic DNA from the biological samples first needs to be sheared by sonication into small enough fragments for subsequent denaturation, primer extension, and addition of the T-Adapters. Random fragmentation of DNA by sonication does not depend on specific DNA sequences, so that the unique ends at random DNA breaks allow for detection of NGS reads coming from independent ligations to the T-Adapter which ensures reliable detection of L1H integration sites. For this reason, we do not use restriction enzymes or other sequence-dependent nucleases techniques.
NOTE: The protocol below was developed using the Covaris E-series sonicator. Other sonicators or mechanical techniques (e.g., shearing through a syringe) may be used to generate randomly-sheared DNA in the size range of 750-1200 bp. DNA shearing conditions need to be optimized for the specific instrument used to generate fragments of 750-1200 bp. Regardless of the instrument used for this step, fragmentation must be random. Do not use nucleases or other enzymatic approaches for the reasons noted above.
For each DNA sample to be sequenced, prepare one library.
- Dilute 1 μg of high-quality genomic DNA (OD 260/280 ratio within a 1.8 to 2.0 range) with 1X Low TE Buffer (10:0.1) in a 1.5-ml LoBind (or similar) tube to a total volume of 50 μl.
- Randomly fragment DNA to a size range of 750-1200 bp. For the Covaris E-series or S-series instruments, follow the manufacturer’s instructions. An example of parameter settings from the Covaris E instrument are the following:
Parameters Settings Duty factor 0.2 Cycles per Burst 200 Treatment Time 6 minutes Bath Temperature 4 – 8° C
- Slowly transfer sheared DNA into a PCR tube or well of the 96-well plate.
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