Chipmentation for HSCs and HPCs


  1. Stain and sort 10,000 – 50,000 HSCs or HPCs in 1.5 mL tube. Spin and pellet in swinging bucket centrifuge.
  2. Resuspend in 150 µL PBS/10%FCS. Add 10 µL of 16% formaldehyde (Pierce, Thermo #28906). Incubate on rotator for 10 minutes at room temperature.
  3. Add 24 µL 1M glycine. Rock 5 minutes at room temp.
  4. Centrifuge in swinging bucket centrifuge. Remove supernatant and wash with cold PBS + 1 µM PMSF + 5 mM Na-butyrate.
  5. Flash freeze in liquid nitrogen and store at -80°C.


Sonication with Covaris – E220 and add antibody
  1. Make 1x sonication buffer and 1x equilibration buffer – for 700 µL add 350 µL of 2x sonication or elution buffer, 100 µL of 7X Complete inhibitor cocktail (Roche) and 3.5 µL of 200 mM PMSF.
  2. Resuspend cell pellet in 130 µL of 1x sonication buffer and transfer to Covaris micro tube – snapcap.
  3. Sonicate cells on Covaris E220 – peak incident power = 105; duty factor = 2%; cycles/burst = 200; duration 840 seconds.
  4. After sonication is completed, transfer to 1.5 mL DNA low bind tube and add 195 µL of 1x equilibration buffer (325 µL final volume).
  5. Spin at 14,000 xg in refrigerated microfuge for 10 minutes. Transfer supernatant to another 1.5 mL DNA low bind tube. Reserve 10% input for control (or pool 10 mL from four replicates). Bring to 40 µL final volume with equilibration buffer.
  6. Add 1.5 µg of H3K4me1 or H3K27ac antibody (Diagenode). Incubate overnight on rotator in cold room.
  7. Per IP, remove 10 µL of protein A Dynabeads into 1.5 mL tube and place on magnet. Wash 2x with RIPA-LS buffer.
  8. Remove supernatant and add 1 mL of RIPA-LS + 0.1% BSA (from RIPA-LS 10% BSA stock 1:100).
  9. Incubate beads overnight on rotator in cold room.


  1. Place blocked beads on magnet. Remove all but 10 µL per reaction. Resuspend and add 10 µL to each IP tube and rotate for 2 hours in cold room.
  2. Prepare buffers – for 2.5 mL each of RIPA-LS, RIPA-HS, RIPA-LiCl and 10 mM Tris, pH 8.0, add one quarter of a Complete protease inhibitor tablet. Add PMSF to 1 µM.
  3. While working in cold room, place beads on magnet and remove supernatant. Wash with 150 µL RIPA-LS x2, then RIPA-HS x2, then RIPA-LiCl x2, then 10 mM Tris x1. After each buffer change, rotate tubes on magnet to move beads around.
  4. After final wash, resuspend beads in 150 µL 10 mM Tris, pH 8.
    (Note: for ChIP-qPCR, proceed directly to elution steps).
  5. Prepare 25 µL tagmentation reaction per sample on ice in separate tubes: 12.5 µL 2x tagment buffer, 11.5 µL water, 1 µL tagmentation enzyme (transposase).
  6. Add samples to magnet and discard the supernatant. Add tagmentation reaction mix, resuspend cells and transfer to 37°C waterbath or drybath.
  7. Incubate for 10 min at 37°C, then add 150 µL of RIPA-LS and place on ice.
  8. Wash with 150 µL cold RIPA-LS x2, and then 150 µL TE x2, moving samples as before.
  9. Remove supernatant and add 48 µL ChIP elution buffer followed by 2 µL proteinase K. Transfer to PCR tubes.
  10. For input samples, add 1.8 µL of 5M NaCl, 0.6 µL of SDS and 5.6 µL of 10 mM Tris pH 8 to 40 µL of pooled input sample (salt and SDS equivalent of RIPALS, brought to final concentration of the elution buffer) in a PCR tube. Add 2 µL of proteinase K.
  11. Incubate at 55°C 1 hour, then 65°C for 6-10 hours.
  12. Place on 96 well plate magnet, remove and save supernatant in a new PCR tube.
  13. Add 126 µL of SPRI beads (1.8x) and mix 15x with pipet. Incubate 10 min at room temp.
  14. Place on magnet for 10 minutes and discard supernatant.
  15. Wash with 200 µL 80% EtOH x2.
  16. Air dry 5 minutes on magnet.
  17. Remove from magnet and add 22 µL buffer EB (10 mM Tris) for experimental samples or 11.5 µL of buffer EB for input. Incubate 10 min.
  18. Remove and save supernatant in 1.5 mL DNA low bind tube. Freeze experimental samples until library amplification. Tagment input as in step 19 – this can be done on day 3 if necessary.
  19. Add 12.5 µL of 2x tagmentation buffer and 1 µL of Tn5 to the input sample. Incubate at 55°C for 5 minutes. Purify over a Qiagen minielute column and elute with 22 µL buffer EB.


  1. Perform qPCR to determine cycle count for library amplification: Master mix with 2x NebNext HF – per 20 µL reaction 0.5 µL each primer, 0.1 µL 100x SYBR, 10 µL NebNext, 7 µL water (make 1.2x necessary volume). Add 18 µL master mix to 2 µL Chipmentation DNA and perform qPCR. (72°C 5 min; 98C 30 sec; (98°C 10 sec, 63°C 30 sec, 72°C 30 sec)x25; 72°C 1min, hold).
  2. Add “+1” to the Ct counts for optimum library amplification.
  3. Prepare Library PCR: 1.5 µL each primer (index + common), 25 µL 2x NebNext HF, 18 µL Chipmentation DNA, 4 µL water. PCR (72°C 5 min; 98°C 30 sec; (98°C 10 sec, 63°C 30 sec, 72°C 30 sec xCt+1 cycles; 72°C 1min, hold at 10°C).
  4. Add 90 µL (1.8x) Ampure SPRI beads. Mix with pipette. Incubate 10 minutes. Use the 96 well plate magnet for this step.
  5. Wash beads on magnet 2x with 200 µL of 80% EtOH.
  6. Air dry and resuspend in 50 µL water. Incubate 10 min, remove beads and save eluate. Ok to freeze o/n at this point.
  7. Add 32.5 µL Ampure beads to eluate from step 6 (0.65x). Mix and incubate 10 minutes. Then place tubes on magnet.
  8. Transfer supernatant to a fresh PCR tube and add 12.5 µL beads (0.9x final – 0.65 + 0.25). Incubate 10 minutes and add to magnet.
  9. Remove supernatant and discard. Wash beads 2x 100 µL with 80% EtOH.
  10. Air dry beads and resuspend in 15 µL water.
  11. Remove beads with magnet. Transfer eluted library to a fresh labeled 1.5mL Low-bind tube and send to GTAC for sequencing