ChIP2 protocol (modified from Rick Young’s protocol-Whitehead Institute)

 

1.      Start with 5x107 cells in a 150 cm2 dish per time point, although we have scaled ChIP down to 10cm2 and dishes and even 6 well format and this works fine.

 

For MCF7 cells. Start with a 15 cm dish at 90% confluence in red medium. Split into 4 15 cm dish into white medium. Change medium every two days and induce on the fourth day. The extract can be used for 2-3 independent IPs. Use 2 ul from a final elution in 60 ul per QPCR of 40 cycles.

 

The day of induction

2.      Prepare antibody-Dynal magnetic beads. (Wash 10 ul of each A/G Dynal beads, per sample, three times in 500 ul of cold PBS plus BSA (5mg/ml) and incubate with your antibody in cold PBS plus BSA (5mg/ml) for at least 6 hours with 500 ul PBS plus BSA (5mg/ml) on the mixer at 4oC. Wash beads twice with cold PBS plus BSA (5mg/ml), resuspend in Dilution buffer and add to chromatin on step 11)

3.      Induce cells by adding ligand to medium (E2 = 1x10-8M) for required time (standard = 45 min)

4.      Prepare 1% formaldehyde (fisher F79-500) to fresh media without serum and warm to 37oC. For 50 ml add 1.35 ml of 37% formaldehyde solution. Replace media with the warmed media containing the formaldehyde and leave for 10 min in incubator. For 10 cm plate use 10ml, for 15 cm plate use 15ml.

5.      Remove media and wash the cells (on the plate) 1x with cold PBS plus BSA (5mg/ml) and 1x with cold PBS.

6.      Scrap into 350ul for 10cm plate, 500ul for 15cm plate, of ice cold PBS complete (PBS containing protease inhibitors) (Complete caplets from roche, 1 caplet in 1ml = 100X concentration)

7.      Spin cells immediately at 2000 rpm for 1 min

8.      Remove PBS complete and flick the pellet to resuspend in residual PBS. Add 350ul of lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH8.1 plus protease inhibitors)…make up fresh. (You can freeze at this step to –80oC, 1st in liquid nitrogen)

9.      Sonicate 3 x 10 sec. Keep tip of sonicator just below the surface of the lysis buffer. The setting is dependent on the specific sonicator and will need to be optimized beforehand by trying several sonication conditions on fixed input DNA, reversing the cross-linking and running the sonicated DNA on gel. With current sonicator use 12% amplification.

10.  Spin 15 min at max speed at 4oC. You should have a pellet of ~20ul. Supernatant is the total chromatin used for PCR later as input. Sample can be freezed at –80oC for weeks. Take a small sample (12 ul from 350 ul (~5%) as input and leave in freezer). Remember to reverse formaldehyde fixing on input as well as sample (Step 16). Also take 5 ul/plate to put on a gel to verify the sonication step, expect 500-1500bp smear.

11.  Dilute sample in dilution buffer (1% triton, 2mM EDTA, 150mM NaCl, 20mM Tris-HCl pH 8.1). Try to dilute at least 1:5 if you can 1:10

12.  Add A/G Dynal magnetic beads prebound to antibody of choice. Start IP as normal O/N 4oC.

13.  Collect magnetic beads using a magnetic concentrator and wash 6 times in RIPA buffer (50mM HEPES pH 7.6, 1mM EDTA, 0.7% Na Deoxycholate, 1% NP-40, 0.5M LiCl). Incubate on mixer for 20 min between every second wash.

14.  Wash twice in 1 x TE (pH 7.6)

15.  Remove all residual TE after last wash

16.  Add 100ul of 1% SDS and 0.1M NaHCO3. Due the same to Input samples. Vortex beads in this solution every few minutes for 30 min total. While at 65oC O/N to reverse cross-linking. Minimum of 6 hours reverse, but a maximum of 14-16 hours.

17.  Purify with QIAquick spin kit (PCR purification kit)- add 500 ul PB, vortex, collect beads, transfer supernatant to column, spin add 500 ul PE, spin, 500 ul PE, spin, spin, collect in 50 ul EB buffer for IP and 100 ul for Input. Spin, Add 75 ul to IP tubes and 150 ul to Input tubes. Quantify using picogreen

18.  Use 5ul for real-time PCR (You may be able to dilute this sample depending on your ChIPing efficiency. If it does not work at these concentration change Abs).

5 ul DNA

4 ul H2O

1 ul Primers (5uM each)

10 ul 2X SYBR

 

For ChIP-Chip

Rnase and Proteinase K treatment

19.  Add equal amoints of material from IPed DNA and input. Use between 1 and 2 ng of total DNA

20.  Make Volume to 200 ul in TE pH 7.5 and add RNase (Sigma R5500) to a final concentration of 0.2ug/ul. Incubate at 37oC for 1-2 hours.

21.  Add proteinase K (Roche, cat # 03-115-836-001) to a final concentration of 0.2ug/ul and incubate at 55oC for 2 hours.

22.  Extract once with equal volumes (200ul) of phenol:chloroform:IAA (Ambion/ Applied biosystems) vortex 20 sec. Prepare phase lock gels (Eppendorf/ Brinkmann Instrument Inc, order no. 0032007.961/cat#955154169). Quick spin phase lock gels, add samples on top without mixing. Spin 5 min 14 000 rpm rt. Transfert top layer to new tube (BE VERY CAREFUL NOT TO TOUCH THE MIDLE PHASE, IT’S BEST TO TAKE LESS).

23.  Add 30ug of glycogen (invitrogen cat # 1286677) and 1:10 volume (20ul) of 3M NaAc.

24.  Add 2 x volume of 100% EtOH and incubate 30 min –80oC.

25.  Spin max speed for 15 min and wash in 150 ul of 70 % EtOH.

26.  Spin max 10 min and air dry pellet briefly.

27.  Resuspend in 17 ul of dH2O.

 

End Filling and blunt ended ligation

1)      On ice add:

2.5 ul 10x end-it buffer

2.5 ul End-it ATP (10mM)

2.5 ul End-it dNTP (2.5mM)

0.5 ul End-it Enzyme Mix

2)      Incubate at r.t. for 30 min

3)      Add 75 ul H2O, Add 1:10 Volume of 3M NaAc pH 5.2 (10 ul) and 10 ug glycogen

4)      Extract once with phenol:chloroform:IAA using phase lock gels. (SAME RECOMMENDATIONS AS ABOVE)

5)      Add 2 Volume 200 ul 100% EtOH, 30 min –80oC

6)      Spin at max speed 15 min 4oC

7)      Wash with 150 ul of 70% EtOH, spin max speed for 10 min 4oC

8)      Resuspend dried pellet in 3.3 ul dH2O

9)      All on ice (include thawing of ingredients), add:

10 ul 2X NEB quick ligase buffer

6.7 ul of annealed linkers (15 uM) – see below

1 ul Quick ligase (NEB)

3.3 ul of DNA sample

Total 21 ul to each sample

10)  Incubate 16oC O/N or r.t for 30 min

11)  Add 79 ul H2O, Add 1:10 Volume of 3M NaAc (10 ul)

12)  Add 2 Volume of EtOH 100% (220 ul), 30 min at –80oC

13)  Spin max speed 15 min 4oC.

14)  Wash in 150 ul 70% EtOH and spin max speed for 10 min 4oC

15)  Resuspend dried pellet in 25 ul of dH2O for 1 LMPCR

 

Annealing of linkers:

16)  Oligo 102 –GCGGTGACCCGGGAGATCTGAATTC-

Oligo 103 –GAATTCAGATC-

 

Make up : 250 ul Tris-HCl (1M) pH 7.9

375 oligo 102 (40 uM stock)

            375 oligo 103 (40 uM stock)

 

17)  Heat at 95oC for 5 min, then immediately into 70oC heat block (with water in the holes) and remove heat block and leave on the bench to slowly cool. Freeze aliquots

 

Ligation Mediated-PCR

18)  Use 25 ul of the samples, add 25 ul of:

12.75 ul dH2O

            5 ul 10X Thermopol buffer (NEB)

            5 ul 2.5 mM dNTP mix

            1.25 ul of 40 uM oligo (the longer oligo in the linker)

1 ul Amplitaq (Perkin Elmer/ Applied Biosystem, not Amplitaq Gold!)

19)  Place the 50 ul samples in PCR machine and run program:

55oC 4 min

72oC 3 min

95oC 2 min

95oC 30 sec

60oC 30 sec

72oC 1 min

go to step 4, 24 more times

72oC 4 min

4oC forever

 

20)  Clean up with Qiagen PCR kit

21)  Elute in 60 ul of elution buffer

22)  Combine the 3 LM-PCR from the same sample. Take 2 ul and dilute it 1/10 for QPCR (on previously validated sites to check that LM-PCR has not introduced a bias). LM-PCR should generate approximately 3ug per PCR. 2ug is required per array. 7 array constitute the whole human genome thus 14 ug required total.

 

Labeling

On ice USE DNAseI from Affymetrix (get it from core facility part # 900131). Dilute 1ul of DNaseI in 31ul of Tris 10mM pH7.8 in DEPC H2O. For each sample add 2 ul of diluted DNAse. Mix to 5.5ul of NEB buffer #4 and 47.5 ul of DNA (up to 6ug)

23)  Run the program:

37oC 30 min

95oC 15 min

4oC hold

28.  Run 5 ul on a gel to check for good fragmentation. Smear should be between 50bp and 100bp, although a little larger is OK.

24)  On Ice: add

13 ul TdT buffer (Promega)

1ul Biotin (1mM stock Enzolife sciences)

1ul TdT (30U/ul)

50 ul of fragmented DNA

25)  Run the program:

37oC 16 hours

95oC 10 min

4oC hold

26)  Spin briefly and prepare for hybridization

27)  Have fun making sense of the data.