Ren Lab ENCODE in situ HiC Protocol for Tissue

Transcription

1 Ren Lab ENCODE in situ HiC Protocol for Tissue Pulverization, Crosslinking of Tissue Note: Ensure the samples are kept frozen on dry ice throughout pulverization. 1. Pour liquid nitrogen into a mortar and pestle. 2. Remove tissue from tube and place into chilled mortar and pestle with a clean cold spatula. 3. Grind up the sample with liquid nitrogen using the mortar and pestle. 4. Use a clean cold spatula to scoop ground tissue back to the original tube. 5. Place the sample back onto the dry ice. 6. Clean the mortar and pestle with 10% bleach and 70% ethanol before using it for the next sample. 7. Transfer tissue into a 15 ml conical tube using a clean spatula or pipette tip. 8. Add cold 1x PBS to 5 ml. 9. Add 0.5 ml of stock crosslinking buffer (see recipe below) and rotate the tube at room temperature for 20 min. 10. Stop the crosslinking reaction by adding ml 2.5 M glycine to a final concentration of M. 11. Rotate at room temperature for 5 min. 12. Centrifuge samples at low speed (15min at 2000 x g). 13. Decant the supernatant and wash once with cold 1X PBS. 14. Centrifuge at low speed (10 min at 2500 x g). 15. Decant the supernatant. 16. Store tissue at -80 C or proceed with dissociation. Stock Crosslinking Buffer (11% formaldehyde) Reagent Stock Concentration Final Concentration Volume for 5 ml NaCl 5 M 0.1 M 0.1 ml EDTA 0.5 M 1 mm 10 μl EGTA 0.5 M 0.5 mm 5 μl Hepes ph8.0 1 M 50 mm 0.25 ml Formaldehyde 37% 11% 1.5 ml dh2o ml Tissue Dissociation, Nuclei Extraction, and Restriction Enzyme Digestion Lysis Buffer 1: Lysis Buffer w/o Triton-X (*use half to make Lysis Buffer 2) Reagent Initial Concentration Final Concentration Volume added for 70 ml CaCl 2 1 M 5 mm 350 ul MgAc 1 M 3 mm 210 ul EDTA 500 mm 2 mm 280 ul EGTA 500 mm 0.5 mm 70 ul Tris-HCl, ph M 10 mm 700 ul complete Protease 25x 1x 2.80 ml Inhibitor Cocktail (Sigma, Cat: ) DTT 1 M 1mM 70 ul PMSF 0.1 M/mL (1000x) M 70 ul ml 70 ml

2 Lysis Buffer 2: Lysis Buffer w/ 0.4% Triton-X Reagent Initial Concentration Final Concentration Volume added for 35 ml Triton-X % (v/v) 0.40% 1.4 Lysis Buffer 1 *(see above) N/A N/A 33.6 ml 35 ml 1 M Sucrose Solution Reagent Initial Concentration Final Concentration Amount added for 21 ml Sucrose N/A 1 M 7.19 g MgAc 1 M 3 mm 60 ul Tris-HCl, ph M 10 mm 210 ul ml 21 ml 1. Thaw crosslinked tissue samples in a gentlemacs M-Tube (Miltenyl Biotec, ) in cold room. 2. Add 3ml Lysis Buffer 1 to M-Tube. 3. Dissociate tissue using gentlemacs factory setting Protein-M-tube After dissociation, dilute with 3 ml of Lysis Buffer 2, mix and filter mixture through a 40 µm BD-cellstrainer into a new Falcon tube. 5. Pool equal volume of remaining Lysis Buffer 1 and Lysis Buffer 2 to make a new lysis buffer (hereafter Lysis Buffer 3 ) to reach final concentration of 0.2% Triton-X. 6. Wash cell strainer with 2 ml Lysis Buffer Centrifuge: 1000 x g, 5 min (Important: reduce slow down speed to 3) 8. Remove and discard supernatant 9. Resuspend nuclei pellet with 1 ml Lysis Buffer 3 and carefully overlay 3 ml 1M sucrose solution to create a density gradient. 10. Centrifuge: 1000 x g, 5 min (Important: reduce slow down speed to 3) 11. Remove and Discard supernatant. 12. Gently resuspend pellet in 50uL of 0.5% SDS and incubate for 10 min at 62 C. 13. Add 145 ul water and 25uL of 10% TritonX-100. Mix well, avoid bubbles. Incubate for 15 min at 37 C. 14. Add 25 ul of 10X NEB2 and 100 U of MboI and digest chromatin overnight at 37 C at 700rpm rotation. Biotin Fill-in, Proximity Ligation, Crosslink Reversal 1. QC: Check digestion efficiency on agarose gel: a. To reverse crosslink a test aliquot, incubate the following at 60 C for 1 hr.: i. 77 ul 10 mm Tris-HCl ph 8.0 ii. 10 ul of 10% SDS iii. 5 ul Proteinase K (10 mg/ml) iv. 8 ul digested sample v. Volume = 100uL b. After incubation, purify using 1 x AmpureXP Clean Up, elute in 30uL of 10mM Tris-HCl ph 8. c. Run 15 ul on 0.8% agarose gel at 190V for 40 min. Save the 15 ul for comparison with post-ligation products. 2. Inactivate MboI by incubating sample at 62 C for 20 min. Then cool to RT for 10 min. 3. To biotin label fragmented ends add the following to the sample: a. 37.5uL of 0.4nM biotin-14-datp (Life Tech, ) a. 1.5ul of 10 mm dctp b. 1.5ul of 10 mm dttp

3 c. 1.5ul of 10 mm dgtp d. 8 ul of 5U/ul Klenow (NEB, M0210) e. = 50 ul 4. Mix by pipetting and incubate for 90 min at 37 C with rotation (500rpm). 5. To ligate, add the following to the sample in the order listed: f. 663uL of H2O g. 120uL of 10X T4 DNA ligase buffer (NEB, B0202) h. 100uL of 10% TritionX-100 i. 12uL of 10mg/mL BSA j. 5uL of 400U/uL T4 DNA Ligase (NEB, M0202) 6. Mix by inversion and incubate at RT for 4 hrs. with rotation (300rpm). 7. To reverse crosslink, add the following to the sample: k. 50uL of 20mg/mL Proteinase K (NEB, P8102) l. 120uL of 10% SDS 8. Mix by inversion and incubate for 30 min at 55 C. 9. Add 130uL of 5M NaCl and incubate overnight at 65 C. DNA Purification, DNA Sonication, Size Selection 1. Cool tubes to RT. 2. Split sample into 2 x 720 ul aliquots in 2 ml microfuge tubes. 3. Add the following to ethanol precipitate the DNA: a. 1.6 X volume of cold 100% ethanol b. 0.1 X volume of 3M Sodium Acetate, ph Mix by inversion and incubate for at least 30 min at -80 C. 5. Centrifuge at max speed for 15 min at 4 C. Remove supernatant and keep on ice. 6. Resuspend one pellet in 800uL of 70% ethanol, resuspend the other pellet with the same mixture and transfer everything to a new 1.7ml tube. Spin down at max speed for 5min at 4 C. Remove supernatant. 7. Wash pellet once more with 800uL of 70% Ethanol. Spin down again. Remove supernatant by pipetting. Air dry pellet for 10 min at RT. 8. Dissolve pellet in 130uL of 1X Tris Buffer (10mM Tris-HCl, ph 8.0) and incubate for 15min at 37 C to dissolve DNA. 9. QC: Check ligation efficiency by gel. 10. Proceed to DNA sonication using Covaris M220: a. Shear DNA in a microtube-130 using the following parameters: i. Peak power: 30.0 ii. Duty Factor: 10.0 iii. Cycles/Burst: 200 iv. Duration: 60s 11. After shearing, bring up the volume to 200 ul with. 12. To remove 500 bp + fragments, add 115 ul SPRI beads (0.55x) and incubate for 5 min at RT. 13. Place on magnet to collect beads, and transfer the supernatant to a fresh tube. 14. Perform a second size selection, add 85 ul of SPRI beads to the fresh tube. Incubate for 5 min at RT. 15. Wash twice with 700 ul of 70% ethanol. Air dry for 5 min at RT. Elute in 300 ul 10 mm Tris-HCl Check size selection on a 2% agarose gel (130V, 45 min). Biotin Pulldown 1 x Tween Wash Buffer (1x TWB) Reagent Initial Concentration Final Concentration Volume added for 10 ml Tris-HCl ph M 5 mm 50 ul EDTA 0.5 M 0.5 mm 10 ul NaCl 5 M 1 M 2 ml Tween-20 10% 0.05% 50 ul

4 7.89 ml 10 ml 2 x Binding Buffer (2x BB) Reagent Initial Concentration Final Concentration Volume added for 5 ml Tris-HCl ph M 10 mm 50 ul EDTA 0.5 M 1 mm 10 ul NaCl 5 M 2 M 2 ml Molecular Biology Gade 2.94 ml Water 5 ml 1. Add 100 ul of 10 mg/ml Dynabeads My One T1 Streptavidin to a clean 1.5mL microfuge tube and wash the beads by adding 400uL of 1X Tween Wash Buffer. 2. Wash thoroughly by mixing up and down with pipet. Separate on magnet and discard the supernatant. 3. Resuspend the beads in 300 ul of 2X BB, then transfer the beads to the sample tube. Incubate the biotin pulldown for 15 min at RT. 4. Separate on a magnet and discard supernatant. 5. Wash beads by adding 600 ul of 1X TWB and transfer to a fresh tube. Heat on thermomixer for 2 min at 55 C with mixing at 350 rpm. Then bind beads to magnet and remove supernatant. 6. Repeat wash in Step 5 at 55 C. 7. Resuspend beads in 100 ul 1X NEB T4 DNA ligase buffer (NEB, B0202) and transfer to a new tube. Bind beads to magnet and discard the supernatant. 8. To repair fragmented ends and remove biotin from unligated ends, resuspend the beads in 100 ul of the following: a. 88uL 1X NEB T4 DNA ligase buffer (NEB, B0202) b. 2uL of 25mM dntp mix c. 5uL of 10U/ul NEB T4 PNK (NEB, M0201) d. 4uL of 3U/ul NEB T4 DNA Polymerase (NEB, M0203) e. 1uL of 5U/ul Klenow (NEB, M0210) f. Volume = 100uL + beads 9. Incubate for 30 min at RT. Separate on a magnet and discard the solution. 10. Wash by adding 600 ul 1X TWB and transfer to a new tube. Heat tube for 2 min at 55 C with mixing. Place 11. Repeat previous step. Discard supernatant. 12. Resuspend beads in 100 ul 1X NEB Buffer2 and transfer to a new tube. Place on magnet and discard supernatant. 13. To add da-tail, resuspend the beads in 100 ul of the following: a. 90 ul of 1X NEB Buffer2 b. 5 ul of 10mM datp c. 5 ul of 5U/ul Klenow (exonuclease) (NEB, M0212) d. Volume = 100 ul + beads 14. Incubate for 30 min at 37 C. Separate on a magnet and discard the supernatant. 15. Wash by adding 600 ul 1X TWB and transfer to a new tube. Heat tube for 2 min at 55 C with mixing. Place 16. Repeat previous step. Discard supernatant. 17. Resuspend beads in 100 ul 1X NEB Quick Ligation Reaction Buffer (NEB, B2200S) and transfer to a new tube. Place on a magnet and discard the supernatant. 18. Resuspend beads in 50 ul of 1X NEB Quick Ligation Reaction Buffer. 19. To ligate adapters by adding the following: a. 2 ul of NEB DNA Quick Ligase (NEB, M2200)

5 b. 3 ul of Illumina Indexed adapter 20. Mix thoroughly and incubate for 15 min at RT. Separate on magnet and discard supernatant. 21. Wash by adding 600 ul 1X TWB and transfer to a new tube. Heat tube for 2min at 55 C with mixing. Place 22. Repeat previous step. Discard supernatant. 23. Resuspend beads in 100 ul of 1X Tris Buffer and transfer to a new tube. Place sample on magnet and discard supernatant. 24. Resuspend beads in 50 ul of 1X Tris Buffer. Store at -20 C until library prep. Library Prep 1. Run a KAPA qpcr assay to estimate concentration and cycle number (n) needed to reach final amount of 20 nm. 2. Perform PCR using Phusion Polymerase (Thermo). Set up reactions as follows: Reagent Volume per reaction T1 bound DNA in 1x Tris 11 ul dntp (10 mm) 1 ul Phusion Polymerase 0.5 ul Forward primer (10 um) ul Reverse primer (10 um) ul ul 50 ul Step 1: 95 C for 30 sec Step 2: 95 C for 10 sec Step 3: 65 C 30 sec Step 4: 72 C for 30 sec Step 5: repeat step 2 4 (n-1) times Step 6: 4 C on hold forever 3. Perform 1 x AMPure Cleanup, elute in 25 ul 10 mm Tris-HCl ph 8.0.