NextSeq 500 paired end sequencing; Genome-wide identification and DNA methylation of human full-length LINE-1 (L1) retrotransposons in a panel of embryonic stem cells, induced-pluripotent stem cells and their progenitor cells
Genome-wide identification and DNA methylation of human full-length LINE-1 (L1) retrotransposons in a panel of embryonic stem cells, induced-pluripotent stem cells and their progenitor cells
Cells were grown and collected from culture dishes. HFF-1 and derivatives were transduced by the Sleeping Beauty transposon vector, described in Grabunzija et al. 2013 (doi: 10.1093/nar/gks1305). The PEli003-A cells were reprogrammed with CytoTune®-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific) supplying the four Yamanaka factors, OCT4, SOX2, KLF4 and c-MYC. The other cell lines were transduced with lentivirus vestors as described in Haase et al. 2009 (doi: 10.1016/j.stem.2009.08.021). Genomic DNA was extracted and prepared using QIAGEN DNeasy Blood & Tissue Kit, according to manufacturer's protocol In brief, bs-ATLAS-seq follows these steps: fragmentation of genomic DNA by sonication, ligation of a methylated adapter, bisulfite treatment of linker-ligated DNA, suppression PCR-amplification of L1 5' junctions, and asymmetric Illumina paired-end sequencing (90 bp + 210 bp). Read #1 starts from a random position (sonicated DNA break), while read #2 starts from a fixed sequence in L1 5' UTR (internal promoter). With this design, read #2 covers the 15 most 5' CpG sites (1-207, relative to L1HS consensus sequence). Read #1 has the same orientation as the L1 element.