Single Nuclei Isolation Protocol

Buffers:

  1. Nuclear Isolation Buffer (NIB)
  2. 400ul 1M Tris-HCl pH=7.4
  3. 80ul 5M NaCl
  4. 120ul 1M MgCl2
  5. 400ul 10\% NP40
  6. 39mls sterile H2O
  7. Nuclear Wash Buffer (NWB)
  8. 1x PBS
  9. 0.5\% BSA
  10. 0.2U/ul Rnase Inhibitor
  11. 1M Tris-HCl pH=7.5Invitrogen - Thermo FisherCatalog \#15567-027
  12. 5M NaClAmbionCatalog \#AM9760G
  13. 1M MgCl2AmbionCatalog \#AM9530G
  14. Surfact-Amps NP-40Thermo Fisher ScientificCatalog \#28324
  15. Protector RNase InhibitorSigma AldrichCatalog \#03335399001
  16. Albumin, Bovine Serum, 10% Aqueous Solution, Nuclease-FreeMillipore SigmaCatalog #126615-25ML
  17. UltraPure Distilled Water Invitrogen - Thermo FisherCatalog #10977-015
  18. 10x PBSThermo Fisher ScientificCatalog #AM9624

Tissue Douncing Preparation

  1. Place homogenizer on ice in preparation for nuclei isolation.
  2. Cut away 40g of tissue.
  3. Mince 10g of tissue at a time into fine (~2mm x ~2mm) pieces. Tissue is minced in portions to avoid lipolysis of adipocytes that will occur if it is all minced together initially.
  4. Place tissue into 15ml dounce homogenizer- FisherScientific K8853000-0015 15 ml Kimble Kontes Dounce Tissue Grinders

Isolate Nuclei

  1. Add 10ml of NIB to homogenizer.
  2. Slowly push down the tissue with the A piston
  3. Run piston through the tissue 10-15, until the piston can smoothly move up and down.
  4. Use a 10ml pipette to transfer the fat/nuclei suspension from the dounce homogenizer into a 50ml conical through a 70uM mesh filter. Change filter if it becomes clogged.
  5. Repeat steps 2 and 3 three times until all of the tissue has been homogenized in 10g portions.
  6. °C Steps should generally be performed on ice as to preserve the integrity of nuclei.
  7. Nuclei should be inspected by bright field before FACS purification. Note: there will be significant debris.

Wash and Stain Nuclei

  1. Centrifuge Nuclei/Tissue suspension at 500xg for 5 mins at 4C.
  2. Remove supernatant carefully. There will be a lipid layer supernatant, it is important to remove this layer first, as removing the subnatant first can result in a lipid/nuclei suspension that is difficult to separate.
  3. Resuspend nuclei pellet in 1ml of NWB and strain through 35uM filter into a FACS tube.
  4. Centrifuge nuclei in NWB at 500xg for 5mins at 4C.
  5. Remove supernatant and resuspend nuclei pellet in NWB with 10ug/ml of Hoechst.
  6. Nuclei are ready for FACS purification.

FACS Purification of Nuclei

  1. Use Corning 352235 FACS tubes
  2. Centrifugation should be at 4 degrees C
  3. Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in WaterInvitrogen - Thermo FisherCatalog #H3570

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