SOP: Nuclei isolation from tissue and DNaseI treatment Date modified: 090923 Modified by: P. Sabo. (UW) The following protocol describes the isolation of nuclei from tissue. Ordering Information Item. Catalog No Manufacturer Calcium Chloride 1M 21115 Fluka EDTA 0.5 M 1 Liter 9262 Ambion EGTA 0.5M ph 8.0 100 ml NC9118216 Fisher Scientific IGEPAL CA-630 I8896-50ML Sigma-Aldrich NaCl 5M solution 500 ml 46-032-CV Mediatech Inc. Potassium Chloride P4504 Sigma-Aldrich Proteinase K >800 u/ml P4850 Sigma-Aldrich Ribonuclease A R4642 Sigma-Aldrich Deoxyribonuclease I D4527 Sigma-Aldrich SDS 10% Solution 9823 Ambion Spermine Free Base 0215207001 MP Biomedicals Inc Spermidine Free Base 0215206801 MP Biomedicals Inc Tris-HCl 1M ph 8.0 46-031-CM Mediatech Inc. Sucrose BP220-1 Fisher Scientific Tricine T5816 Sigma Aldrich MgCl2 M1028 Sigma Aldrich Optiprep 89049-128 VWR Complete protease Inhibitor 04693132001 Roche Applied Science Milli-Q or Molecular Biology Grade Water Materials List 0.2 um Filter bottles (500, 1000 ml) Micropipet w/ P20 tips Micropipet w/ P200 tips Micropipet w/ P1000 tips Graduated pipets (5, 10, 25 ml) 15 ml and 50 ml Conical Tubes Dounce 7ml Tissue Grinder (22877-280 VWR) Hemocytometer Microscope (preferably phase contrast)
I. Nuclei Prep Prior to Nuclei Isolation: 1. Prepare homogenization buffers, add proteinase inhibitor. Keep on ice. 2. Add spermine, and spermidine to 1X Stop Buffer. (if SDS has precipitated out of solution warm to 37C to re suspend SDS prior to adding supplements) 3. Prepare fresh 1X DNaseI Buffer (Dilute 10X DNaseI buffer 1:10 with BufferA) 4. Aliquot DNaseI Buffer: 50ml conical, 5-10ml DNaseI Buffer (1ml per 10.0 E+6 expected nuclei). or 15 ml conical, 1-5 ml DNaseI Buffer (1ml per 10.0 E+6 expected nuclei). 4. Warm Stop Buffer and DNaseI Buffer (minus DNaseI) in 37 o C water bath. Allow to equilibrate for 60 minutes prior to use. 5. Pre-chill centrifuge to 4C. All centrifugation should be done at 4C. Estimated buffer amounts per tissue: Mg Homogenization Solution: EDTA Homogenization Buffer: Optiprep Dilution Buffer 10 ml 20 ml 1.0 ml 50 % optiprep Solution 6.0 ml Buffer A Stop Buffer 20 ml 5.0 ml 2 Percent Formaldehyde soln 5.0 ml 1M Glycine 1.5 ml 2 dounce homogenizers 1 reusable filter unit 120 um mesh 1 reusable or disposable filter unit 40 um mesh.
Nuclei Isolation from solid tissues Tissue received should be 1 sq cm or smaller in size in UW Belzer solution All solutions should be kept on wet ice. 1. Add 5 mls Mg homogenization solution to dounce on ice. 2. Remove tissue from tube (approx 1 sq cm) and cut into ~2 mm square pieces and transfer to dounce. 3. Slowly dounce with loose fitting pestle 5-10 times. Tissue should be reduced to ~90% homogenous small particles. May require more for fibrous tissue. 4. Filter solution through 120 um filter into second dounce to remove large particulates. 5. Bring volume back to 5 ml. 6. Slowly dounce with tight fitting pestle 5-10 times slowly depending on tissue consistency. 7. Filter solution through 40 um filter. 8. Bring volume to 5 mls with homogenization solution. 9. Add 3 mls of 50% Optiprep soln and mix by inverting until homogenous. (approximately 10 times) 10. Layer onto 3 ml 25% 2 ml 35% two step Optiprep gradient. (Percent of upper level may have to be adjusted depending on tissue type. Determine empirically. Use Falcon 2059 tubes or other 15 ml tubes rated for this. 11. Centrifuge 20 minutes at 6100xg in swinging bucket rotor using Beckman Avanti centrifuge or equivalent. 12. Upon removal from centrifuge nuclei should be visible at the 25-35% interface. 13. Aspirate off upper layer of homogenate and 1 ml of 25% layer. 14. Transfer nuclei at 25-35% interface to clean 15 ml conical containing 12 ml s EDTA homogenization buffer on ice. Approximately 2ml s will be transferred. 15. Mix gently and take 10 ul for nuclei count. 16. Pellet nuclei by centrifugation for 10 minutes at 250-500 xg. (May be optimized for specific tissue) 17. Aspirate off supernatant and resuspend nuclei in 10 ml s buffer A. 18. Aliquot into 15 ml conical treatment tubes based on nuclei count in step 15. Optimal 10 million nuclei. Minimum 5 million. 19. Pellet nuclei by centrifugation at 250 xg for 5 minutes. 20. Aspirate off buffer and Gently tap tube to loosen nuclei pellet. place tube on ice. Proceed directly to DNaseI digestion.
Nuclei Isolation from solid tissues for ChIP Tissue received should be 1 sq cm or smaller in size in UW Belzer solution All solutions should be kept on wet ice. 1. Add 5 mls Mg homogenization solution to dounce on ice. 2. Remove tissue from tube (approx 1 sq cm) and cut into ~2 mm square pieces and transfer to dounce. 3. Slowly dounce with loose fitting pestle 5-10 times. Tissue should be reduced to ~90% homogenous small particles. 4. Slowly dounce with tight fitting pestle 5-10 times slowly depending on tissue consistency. 5. Filter solution through 120 um mesh with reusable filter unit 6. Filter through 40 um mesh with disposable filter unit into 50 ml conical tube. 7. Add 5.0 ml of 2 % formaldehyde solution. 8. Cap and mix by inverting. Incubate 10 minutes at room temp on rotisserie or rocker. 9. Add 1.45 ml s of 1.0 M glycine solution to a final concentration of 0.125M and continue mixing 5 minutes at room temp. 10. Centrifuge 5 min at 300 xg at 4C 11. Remove supernatant and resuspend pellet in 3.5 ml s 20 mm tricine buffer ph 7.8. 12. Add 1.5 ml s optiprep soln. 13. Underlayer with 2.0 mls 25% Optiprep soln in tricine buffer. 14. Centrifuge 20 minutes at 6100 xg. 15. Aspirate off supernatant and resuspend pellet in ice cold PBS. 16. Centrifuge 5 minutes at 300 xg at 4C. 17. Aspirate off Supernatant and store at -70C.
DNaseI Treatment 1. Stop Buffer and 1x DNAseI buffer (10x DNAseI buffer diluted 1:10 with Buffer A) should be equilibrated to 37C in water bath prior to starting nuclei isolation. (buffers should be allowed to equilibrate 30-60 minutes at 37C) 2. Add RNaseI to Stop Buffer and mix thoroughly. 3. Add the appropriate amount of DNaseI enzyme to the DNAseI Buffer just prior to treatment (For example: For an 80 unit/ml digestion, add 32 ul of enzyme (10units/ul) to 4 ml of 1x DNAseI Buffer). Mix thoroughly but gently by pipetting with 10 ml pipette. Pipette slowly/gently as enzyme denatures easily. Remaining steps should be timed carefully. 4. Place tubes with loose nuclei pellets in 37C water bath and allow temperature to equilibrate for 1 minute. 5. Gently re-suspend nuclei with 1x DNAseI buffer plus enzyme. Pipette several times gently to ensure homogenous suspension. 6. Incubate for 3 minutes at 37 C. 7. Add equal volume of stop buffer to DNaseI reaction tube, mix by inverting several times and transfer tube to 55 C waterbath. 8. Allow digestion tubes to incubate at 55 C for 15 minutes 9. Dilute proteinase K 1:20 with stop buffer. (Sigma P4850 23 mg/ml, 1100 units/ml) Add 20 ul diluted proteinase K per ml of DNaseI treated nuclei. Mix sample with proteinase K gently by inverting several times. Protease K may be added to stop buffer just prior to adding to DNaseI/nuclei. 10. Digest sample with Proteinase K 1hr at 55 C. 11. Store treated samples at 4 C. Samples have been found to be stable for up to 2 years at 4C.
Notes: Work quickly using reagents maintained at appropriate temperatures. Spin time and force may vary depending on cell type. We find that 5 minutes at 500 xg works well with most mammalian cell lines. For sensitive cells you may have to lower the g-force and or increase the time to 10 minutes. Using DNaseI at 60, 80, 120 units/ml we observe high levels of cutting in HS sites with little cutting in non-hs regions. This difference in cutting can easily be measured using qpcr. This can vary with DNaseI stock and should be verified by empirically.
Stock Reagents: Unless otherwise noted, all buffers and reagents should be filtered (0.22 M) and prechilled to 4 o C (on ice) before use. Stock Solutions 1.0 M CaCl2 As Supplied from Manufacturer 3 M KCl (See Below) 0.5 M EDTA, ph 8.0 As Supplied from Manufacturer 0100 mm EGTA, ph 8.0 As Supplied from Manufacturer 10% IGEPAL CA-630 (See Below) 5 M NaCl As Supplied from Manufacturer 0.5 M Spermidine (See Below) 0.5 M Spermine (See Below) 10 % SDS As Supplied from Manufacturer 1 M Tris-Cl, ph 8.0 As Supplied from Manufacturer Proteinase K >800 u/ml As Supplied from Manufacturer Ribonuclease A As Supplied from Manufacturer RNase-Free DNaseI (10,000 mbu) As Supplied from Manufacturer 1M KCl 250 ml Dissolve 18.64 grams KCL in 250 ml Milli-Q H2O. Autoclave 15 min 121C. Store Room Temp. 0.5 M Spermine Dissolve 5 grams Spermine Free Base in 49.43ml final volume Milli-Q H2O. Store -20C 0.5 M Spermidine Dissolve 1 gram Spermidine Free Base in 13.77 ml final volume Milli-Q H2O Store 4C
DnaseI 10X Digestion Buffer (per 100 ml) Store Room Temp 1 yr. Final concentration Stock concentration Amount used from stock 60 mm CaCl 2 1 M CaCl 2 6 ml 750 mm NaCl 5 M NaCl 15 ml Combine stock solutions, and 79 ml nuclease free, sterile dh20. Stock DNaseI Resuspend DNaseI at 10 u/ul in DNaseI Storage buffer. 20 mm Tris ph 8.0 50 mm NaCl 1mM dithioerythritol 0.1 mg/ml bovine serum albumin 50% glycerol. Aliquot into 50 ul aliquots and store at -20C
Working Buffers Mg Homogenization Buffer (per 50 ml): Final Concentration Stock concentration volume stock soln 25 mm Sucrose 50% sucrose (1.46 M) 0.855 ml 20 mm Tricine ph 7.8 0.5 M Tricine, ph 7.8 2.0 ml 15 mm NaCl 5.0 M NaCl 0.15 ml 60 mm KCl 1.0 M KCl 3.0 ml 2 mm MgCl 1.0 M MgCl 0.1 ml 0.5 mm Spermidine 0.5 M Spermidine 0.05 ml Sterile MilliQ Water 100% 45.8 ml Combine indicated amounts of stock solutions and add sterile dh 2 O to a final volume of 1 Liter. Filter Sterilize.22 um Use within 1 week. Add 1 Complete proteinase inhibitor tablet 1 hr prior to use and allow to resuspend completely. EDTA Homogenization Buffer (per 50 ml): Final Concentration Stock concentration volume stock soln 25 mm Sucrose 50% sucrose (1.46 M) 0.855 ml 20 mm Tricine ph 7.8 0.5 M Tricine, ph 7.8 2.0 ml 15 mm NaCl 5 M NaCl 0.15 ml 60 mm KCl 1 M KCl 3.0 ml 1 mm EDTA, ph 8.0 0.5 M EDTA, ph 8.0 0.1 ml 0.5 mm EGTA, ph 8.0 0.5 M EGTA, ph 8.0 0.05 ml 0.5 mm Spermidine 0.5 M Spermidine 0.05 ml Sterile MilliQ Water 100% 45.8 ml Combine indicated amounts of stock solutions and add sterile dh 2 O to a final volume of 1 Liter. Filter Sterilize.22 um Use within 1 week. Add 1 proteinase inhibitor tablet 1 hr prior to use and allow to resuspend completely.
Optiprep dilution buffer (per 50 ml): Final Concentration Stock concentration volume stock soln Sterile MilliQ Water 100% 17.9 ml 120 mm Tricine ph 7.8 0.5 M Tricine, ph 7.8 12.0 ml 90 mm NaCl 5 M NaCl 0.9 ml 360 mm KCl 1 M KCl 18.0 ml 6 mm EDTA, ph 8.0 0.5 M EDTA, ph 8.0 0.6 ml 3 mm EGTA, ph 8.0 0.5 M EGTA, ph 8.0 0.3 ml 3 mm Spermidine 0.5 M Spermidine 0.3 ml Filter Sterilize.22 um Use within 1 week. 50% Optiprep Solution (per 50 ml): Mix 5 volumes Optiprep with 1 volume Optiprep Dilution Buffer. Mix and store on ice. Buffer A (per Liter): Final Concentration Stock concentration volume stock soln Sterile MilliQ Water 100% 914 ml 15 mm Tris-Cl, ph 8.0 1 M Tris-Cl, ph 8.0 15 ml 15 mm NaCl 5 M NaCl 3 ml 60 mm KCl 1 M KCl 60 ml 1 mm EDTA, ph 8.0 0.5 M EDTA, ph 8.0 2 ml 0.5 mm EGTA, ph 8.0 100 mm EGTA, ph 8.0 5 ml 0.5 mm Spermidine 0.5 M Spermidine 1 ml Combine indicated amounts of stock solutions and add sterile dh 2 O to a final volume of 1 Liter. Use within 1 week. 2X IGEPAL buffer: Buffer A supplemented with IGEPAL at 0.2% final concentration. Make day of use. Add 2 ml 10% IGEPAL stock to 98 ml Buffer A; Keep on Ice The final concentration may have to be adjusted depending on cell line. If you see lysis of nuclei dilute 1:1 with buffer A until there is no observable lysis of nuclei.
1X DnaseI Digestion Buffer Make day of use. For 50 ml add 5 ml 10X DNaseI buffer to 45 ml Buffer A; Allow to equilibrate to 37C for 60 minutes prior to use. Stop Buffer (per Liter) Final concentration Stock concentration Amount used from stock 50 mm Tris-Cl, ph 8.0 1.0 M Tris-Cl, ph 8.0 50.0 ml 100 mm NaCl 5.0 M NaCl 20.0 ml 0.10 % SDS 10% SDS 10.0 ml 100 mm EDTA, ph 8.0 0.5 M EDTA, ph 8.0 200.0 ml Molecular biology grade H20 725.0 ml Combine stock solutions and add sterile dh20 to a final volume of 1 Liter. Dispense into 50-mL aliquots store at 4 C. (SDS will precipitate upon storage at 4C but will go back into solution upon heating to 55 C) On day of use add the following to a 50 ml aliquot: 1) 50 ul 10 mg/ml RNaseA (final concentration 10 ug/ml) 2) 100 ul 0.5 M Spermidine (final concentration 1 mm) 3) 30 ul 0.5 M Spermine (final concentration 0.3 mm) 2.0 Percent Formaldehyde solution 50 ml: Add 2.9 ml 35% formaldehyde to 47.1 ml EDTA Homogenization buffer Store at Room temp.
1 M Glycine solution 50 ml: Final concentration Stock concentration Amount used from stock 1.0 M Glycine 3.76 g Add Molecular biology grade H20 to 50.0 ml 10% IGEPAL CA-630 Stock (per 100 ml): Combine 10 ml 100% IGEPAL CA-630* and 90 ml MilliQ sterile dh20. *100% IGEPAL is extremely viscous; be careful to accurately withdraw 10 ml. 10% IGEPAL should be warmed in a 55 C waterbath for 30 minutes prior to making stock to ensure proper dissolution. Stock has been found stable for 2 years at 4C. Store at 4 C