Figure 1

Overview of the three technologies used in this study (flow cytometry capture, laser capture microdissection, magnetic streptavidin-bead capture). For all three processes, human lymphocytes were cultured overnight, then phytohaemagglutinin (PHA) and colcemid were added to achieve a high mitotic index and accumulation of cells in metaphase. Later, metaphase chromosomes were extracted from the lymphocytes. (A) For flow cytometry capture, the extracted chromosomes were incubated with a specific biotin Y chromosome probe and stained with streptavidin-PE and DAPI. Y chromosomes were then sorted in a FACSAria flow cytometer. The sorted chromosomes were collected in Eppendorf (Hamburg, Germany) tubes in ddH₂O (double-distilled water) for further processing. (B) For laser capture microdissection, individual chromosomes were hybridized with Y chromosome-specific probes conjugated with FITC (green), counterstained with DAPI (blue) and mounted on slides covered by polyethylene membranes. On these slides, they were selected and catapulted by the laser pressure catapulting (LPC) function in a Zeiss PALM MicroBeam IV Laser Microdissector. Y chromosomes were captured within the cap and dissolved in TE buffer. The cap was closed and the sample was spun down by centrifugation. (C) For magnetic streptavidin-bead capture, chromosomes were incubated with a specific biotin Y chromosome probe as in the previous procedure. Dynabeads MyOne streptavidin beads were added to the probe Y chromosome mixture and magnetic separation was performed to capture the Y chromosome on a magnetic rack. Finally, in all cases, physical fragmentation was performed before library prep and sequencing with a Covaris S2 sonicator.