GSM3534633: eccDNA_HT-Control-Rep2; Homo sapiens; OTHER
HT Cell lysate preparation and pull-down on Halo-Link beads: All the reagents used were pre-chilled and the entire procedure was performed on ice. HT-fusion proteins were induced by adding 1 mg/ml doxycycline to cells in a 15-cm plate. Two days later, cells were washed with PBS, scraped and transferred into a micro-centrifuge tube. After centrifugation five packed cell volume (PCV) Hypotonic Buffer was added to cell pellets, allowing the cells to swell, for 15 min on ice. Hypotonic Buffer was removed after spinning. Cells were suspended in 0.5 PCV of Buffer LS and equal volume of Buffer LS with 600 mM NaCl and 0.2% Triton X-100 was added. Cells were homogenized by passing 10 times through a 27G hypodermic needle and rotated for 20 min. After centrifugation, the supernatant was transfer into a new microcentrifuge tube and equal volume of Buffer LS to the supernatant was added. Cell lysate was added to the equilibrated HaloLink Resin and incubated by mixing on a tube rotator for 30 min at room temperature. After centrifugation supernatant was saved as sample flow through. Resin was washed with PD Washing Buffer five times.  The beads were boiled in Laemmli sample buffer to obtain the eluates.
GEO Accession
GSM3534633