Ion Torrent PGM sequencing; Single-cell identification of L1HS-Ta retrotransposons in preimplantation embryos by ATLAS-seq
Single-cell identification of L1HS-Ta retrotransposons in preimplantation embryos by ATLAS-seq
Donated human pre-implantation embryos were thawed at the pre-implantation stage and cultured to the blastocyst stage. Inner cell mass was separated from trophectoderm cells by laser drilling. Single-cells were collected by micromanipulation using a biopsy capillary. Each single cell was transferred into a DNA low-binding tube for whole genome amplification. The remaining cells of the inner cell mass were also collected and treated as an additional sample (remaining embryo, sample named 'T'). Whole Genome Amplification (WGA) was performed by two successive rounds of multiple displacement amplification (MDA) following an approach described by Spits et al. (2006) using the GenomiPhi v2 DNA Amplification Kit (GE Healthcare). 5' ATLAS-seq: The protocol is described in Philippe et al. eLife 2016 (PMID 27016617). Briefly, DNA was randomly sheared by sonication, end-repaired, and A-tailed. After ligating a linker, a suppression PCR step allows amplifying L1HS-Ta1-d 5' junctions and adding Ion Torrent-specific sequencing primers, as well as sample-specific DNA barcodes for multiplexed sequencing.