E-MTAB-6933:R02_SG09_s
Ion Torrent PGM sequencing; Identification of de novo LINE-1 (L1) retrotransposon integration sites in HeLa S3 cells by ATLAS-seq-neo
Identification of de novo LINE-1 (L1) retrotransposon integration sites in HeLa S3 cells by ATLAS-seq-neo
R02_SG09_s
Cells were transfected with a plasmid-borne L1.3 element carrying a NeoR-based retrotransposition cassette and selected by G418 for 10 days. Genomic DNA was extracted using a QiaAmp DNA Blood mini kit (Qiagen) ATLAS-seq-neo libraries were prepared by random mechanical fragmentation of the genomic DNA to ensure high-coverage, ligation of adapter sequences, and suppression PCR-amplification of the 3' end L1 junction with its flanking genomic sequence. The primers used for suppression PCR specifically targets the engineered element and not endogenous copies as in the original ATLAS-seq protocol (Philippe et al. eLife 2016), and are fused to Ion Torrent-specific A and trP1 primers. Libraries are oriented with reads directed from the flanking sequence to the L1 3' end (reads have the following structure: 5'-A-barcode-linker-flank-poly(dT)-L1-trP1-3'). This library construction protocol used suppression PCR reactions performed in solution.
Universite Cote d'Azur, Inserm, CNRS
E-MTAB-6933:R02_SG09_s
Experimental Factor: replicate
4
Experimental Factor: library preparation method
suppression PCR in solution