Alon s SCN ChIP Protocol

Transcription

1 Prior to starting your ChIPs and Shearing 1. Turn on sonifiers and cooling system allow system to reach -1 C before shearing 2. Cool bench top centrifuge to 4 C 3. Prepare all of your buffers with protease inhibitors in advance 4. Keep all buffers on ice. 5. Use DNA low bind tubes throughout protocol (eppendorf cat# ) Preparing Dynabeads ( 1. Make Blocking Buffer +Protease Inhibitors 2. Add 1ml of blocking buffer to each 2ml tube containing 50λ Dynabeads A/G mixture (50/50) 3. Mix by placing tubes on magnet then turning tubes Repeat steps 2 and 3 5. Aspirate blocking buffer and add 200λ of fresh blocking solution to pre washed beads Conjugating Antibody and Magnetic Beads 1. Add antibody of interest to each tube containing 50/50 Protein A/G magnetic beads w/ blocking solution 2. Conjugate ~1µg antibody for active marks (e.g. H3K4me3) and ~5µg for repressed chromatin (e.g. H3K27me3) 3. Rotate for at ~ 2hr at 4 C 4. Make sure that the rotating motion keeps the liquid moving and the beads wet For Frozen Cells Thaw frozen cross linked cells on ice. Make sure that the pellet has thawed completely Cell Lysis If cell number is very low, go right into this lysis and shearing. If cell number is not low, use ~ 100λ of cell Lysis buffer per 1e7 cells max (need to play around with this a bit) 1. Resuspend cross linked cell pelletl in 100λ of Lysis Buffer (LB) + protease inhibitors (pi) 2. Make sure that sample is well resuspended by pipetting 3. Incubate 10 on ice 4. Add 900λ ChIP Dilution Buffer (CDB) plus protease inhibitors to each pellet 5. Proceed to shearing Printed 11/14/2013 6:18:00 PM Page 1

2 Chromatin Shearing using the Branson Sonifier DAY 1 Shearing using the Branson Sonifier can be somewhat subjective. Our set up has a cooling system that uses a glycerol/water mix and is set to -2 C. In addition, a moving platform holds a metal rack that is chilled and keeps tubes in place upon sonication. The following are guidelines only and reflect our current methods. Amplitude varies from one instrument to the next but the energy output in watts will not, therefore we use watts rather than amplitude Settings: On: 0.7 seconds Off: 1.3 seconds Time: 2 minutes Watts: (Alon uses 16 watts) Wait 1-2 between cycles to avoid over heating the samples because they get very hot despite the cooling system (Alon shears 5 with no waiting in between) Shearing: 1. Clean probes with ddh 2 O before and after use 2. Place lysed cells in 1.5 ml eppendorf tube in pre chilled rack that is part of our Branson set up 3. Raise the platform so that the sonifier tip is centered in the tube. 4. Allow the tip to touch the bottom of the tube and then back off the tip so that there is approximately 1-1.5mm of space between the tip and the bottom of the tube. 5. Begin shearing using the settings above for 5 6. Watch the tube for a few seconds to make sure sample is not splashing or foaming 7. After you have completed your shearing remove samples to ice and clean sonifier tip 8. For large cell numbers, place aliquots on ice for at least 10 in pre-chilled and pre labeled tubes 9. After 10 spin lysate for 10 at max 4 C. Transfer supernatant to a new tube leaving any residual debris behind 10. For lower cell numbers, omit steps 8 and Pool all chromatin from the same samples; remove 10λ for reverse cross linking. This is your input material. label tube and store at 4 C or -20 C until reverse crosslinking. This will be your starting material for your whole cell extract library or control library. Printed 11/14/2013 6:18:00 PM Page 2

3 12. Divide the 1ml of chromatin into λ aliquots in small tubes (5-10 ChIPs per sample but you can use more chromatin as well) 13. Add 5M NaCl to your pooled chromatin samples to a final conc. of 167mM a. This will adjust your ChIPs to the same concentration of NaCl as the ChIP Dilution Buffer (CDB) (Omitted in Alon s protocol will wait for seq data to determine whether we should keep or omit this step) Washes b. Combine the 100λ lysate with the 50λ of protein A/G conjugated beads rotating O/N at 4 C In Advance : Prepare thermal cycler for 65 C and have buffers ready so the beads won t dry out 1. Place tubes on magnet a. For low cell numbers, collect all sup to the original tubes used for sonication taking b. Take care to not disturb beads c. This is the low cell number FT- save for reverse C 2. RIPA Buffer washes x6 (DOC (0.1%), 10%SDS (0.1%), Triton X-100 (1%), STE) a. Add 200λ of RIPA, rinse beads to bottom of tube, then transfer beads to 96 well plate b. Save pipet tips from this step in respective tubes in order to transfer remaining beads to 96 well plate 3. Add another 200λ of RIPA buffer to tubes with a clean pipet tip. Use saved tip to collect remaining beads and add them to their respective wells in the 96 well plate 4. Repeat above step 5. Continue with three more RIPA washes in 96 well plate using a multichannel pipet and appropriate magnet 6. Take care not to aspirate any beads 7. Wash x2 with 200λ RIPA/500mM NaCl Buffer (High Salt Wash Buffer) 8. Wash x2 with 200λ LiCl Buffer (0.25M LiCl,1% NP40,1% Na Deoxycholate, 1mM EDTA,10mM Tris-HCl ph 8.1) 9. Wash x2 with TE 10. Elute by adding 50λ ChIP Elution Buffer (10mM Tris-Cl ph 8.0, 5mM EDTA, 300mM NaCl, 0.1% SDS) a. Currently, eluate is removed from bead to reverse cross link 11. Incubate at 65 C 4-6hr or O/N to reverse crosslink 12. Include FT material to plate to reverse cross link Printed 11/14/2013 6:18:00 PM Page 3

4 After reverse cross linking 1. Add 5λ RNAse A and incubate 30 at 37 C 2. Mix by moving on the magnet (use the beads to mix the reaction) 3. Add 5λ Proteinase K, mix as above and incubate 2hr at 37 C 4. Run Gel on 5λ FT, 5λ 100bp ladder (for low cell number ChIPs that where shearing has not been optimized) DNA purification For low cell number ChIPs, go directly to library after clean up 1. SPRI cleanup (use 2.2x SPRI beads or 132λ to ~60λ of eluted and reverse cross linked ChIP material post RNAse A and prot. K treatment 2. Mix *15, 2' RT followed by 4' on magnet 3. Wash x2 with 200λ 70% EtOH for 30s on magnet or until beads completely move to magnet 4. Elute in 40λ 10mM Tris-HCl ph NOTE: For each elution add EB to the top of beads without touching them 6. Cap plates with strip covers or plastic seal vortex quickly and spin down 1000rpm 1 7. For the FT use Qiagen kit, elute in 45ul. Take 5 for gel, use the rest for library prep Qubit measurement 1. Use Qubit dsdna HS setup 2. Make Qubit master mix a. (n+2)*200 λ ( n+2) of Qubit dsdna HyperSensitive Buffer b. (n+2) λ Qubit Reagent (pinkish red, in drawer b/c sensitive to light) 3. In 2 Qubit Assay Tubes a. 10λ Calibration #1, #2 respectively b. 190λ of master mix 4. In remaining n Qubit Assay Tubes a. 2λ DNA b. 198λ master mix 5. Mix well via vortex, quick spin, let stand 1 minute in dark drawer 6. Measure using Qubit Spectrometer and report values in ng/λ a. Calibrate with calibration #1, #2; use #2 as a test sample: expect to get 500 ng/λ Printed 11/14/2013 6:18:00 PM Page 4

5 Running Gel to check Sonication (for low cell numbers) 1. Prepare 1.8-2% Agarose Gel (earlier) a gm/80λ TAE buffer in lightly capped bottle b. Microwave 1-2 minutes, stopping to shake it in between c. Pour into EtBr designated flask and immediately mix 5λ EtBr (0.5µg/λ) d. Cast gel and let cool 30min at room temp, or 7 minutes in -20 C freezer e. Run at 170V 200V in TAE buffer Buffers Chip Dilution Buffer: 0.01% SDS, 1.1% Triton X-100,1.2mM EDTA,16.7mM Tris-HCl ph 8.1,167mM NaCl LiCl Wash Buffer: 0.25M LiCl,1% NP40,1% Na Deoxycholate, 1mM EDTA,10mM Tris-HCl ph 8.1 TE Buffer ph 8.0:10mM Tris-HCl ph8.0,1mm EDTA ph 8.0 ChIP Elution Buffer : 10mM Tris-Cl ph 8.0, 5mM EDTA, 300mM NaCl, 0.1% SDS Blocking Buffer (4 C): PBS, 0.5% TWEEN 20, 0.5% BSA Low Salt Wash Buffer: 0.1% SDS,1% Triton X-100, 2mM EDTA, 20mM Tris-HCl ph 8.1,150mM NaCl RIPA/140mM NaCl Buffer : 20mM Tris-HCl ph 8.1, 1mM EDTA,140mM NaCl,Triton X-100, 0.1%SDS, 0.1% DOC High Salt Wash Buffer: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl ph 8.1, 500mM NaCl RIPA/500mM NaCl Buffer: 20mM Tris-HCl ph 8.1, 1mM EDTA,500mM NaCl,Triton X-100, 0.1%SDS, 0.1% DOC Printed 11/14/2013 6:18:00 PM Page 5