Formaldehyde Crosslinking [ download protocol ]

  1. Grow adherent cells on 15-cm plates. Before crosslinking, carefully split and count one plate. Note: Accurate cell counts are critical for maintaining consistency between cell and lysate batches because cell numbers affect the efficiency of the sonication and DNase treatment during lysis. We typically harvest and crosslink 100-200 million cells in parallel and freeze multiple 20-million cell pellets; one of these pellets is spent to optimize lysis conditions, and the rest are used for purification experiments.
  2. Heat one aliquot PBS at 37 °C and chill one aliquot at 4 °C (see below for volumes).
  3. Remove media from cells. Rinse cells in plate with 10 mL room temperature PBS. Discard PBS.
  4. Immediately before using, prepare a 2% formaldehyde solution in PBS preheated to 37 °C. Use a fresh ampule of 16% formaldehyde (Pierce).
  5. Add 7 mL warmed 2% formaldehyde solution to cells. Incubate at 37 °C for 10 minutes, gently rocking by hand every 3 minutes.
  6. Quench formaldehyde crosslinking by adding glycine to a final concentration of 500 mM. Incubate at 37 °C for 5 minutes.
  7. Discard formaldehyde waste in appropriate disposal container.
  8. Rinse cells three times with cold PBS. Avoid dislodging cells from plate.
  9. After last wash, add 2 mL of ice-cold Scraping Buffer to each 15-cm plate. From this point, keep cells at 4 °C.
  10. Scrape cells from plate and transfer to a 15-mL Falcon tube.
  11. Centrifuge at 1000× g at 4°C for 5 minutes to pellet cells.
  12. Discard supernatant and resuspend pellet in 1 mL ice-cold Scraping Buffer to break up the pellet. Add more Scraping Buffer if necessary for convenient aliquoting (e.g., add 1 mL of Scraping Buffer for every 20 million cells).
  13. Aliquot cells into microcentrifuge tubes (20 million cells each) and spin at 2,000× g at 4 °C for 5 minutes.
  14. Remove supernatant and flash freeze pellet in liquid nitrogen.  Store until cell lysis at -80°C.