Illumina fragment Tn-Seq sequencing of LINE-1 retrotransposons and SNPS in sample MS13212from multiethnic Asian population study.
Briefly, we linearly amplified 3??? flanking regions of L1Hs elements using an L1Hs-specific primer followed by a hemi-specific PCR using a set of eight degenerate primers. We made dilutions of the hemi-specific PCR reactions and used them in a second round of PCR which amplified the library and incorporated the appropriate Illumina sequencing adapters. After the PCR, we ran the products on a 2% TAE agarose gel and excised the fragments between 200 and 500 bp. We gel-extracted the DNA and pooled the eluted DNA from the eight reactions and further column-purified it. We next removed adenine overhangs by incubating with Pfu Turbo DNA polymerase. Finally, we purified the blunt-ended DNA and analyzed it using the High Sensitivity Chip on the Agilent 2100 BioAnalyzer to determine the DNA concentration and quality (protocol modified from Ewing and Kazazian, Genome Research 2010).