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DNA ChIP Protocol

Author: Levy Lab, updated date: , view: 102, Q&A: 0
Tags: DNA, chromatin and immunoprecipitation

Procedure

Dan Levy's Lab 2018
Zlata Vershinin

DNA ChIP Protocol

Combined protocols of

  1. Ainbinder, E., Revach, M., Wolstein, O., Moshonov, S., Diamant, N., and   Dikstein, R. (2002) Mol. Cell. Biol. 22, 6354-6362

  2. Capucine Van Rechem, Stanford University


Crosslinking and chromatin isolation1

  1. Fix cells by addition of Formaldehyde to the medium to a final concentration of 1.5% (400 µl for 10 plate from 37% stock formaldehyde) and incubate 10 min at room temp. (Time may vary and need to be calibrated for each cell line)

  2. Stop crosslinking reaction by addition of Glycine pH 8.5 to a final concentration of 125mM (1ml from 1.25M pH 8.5 Glycine stock). 5 min at room temp.

  3. Discard sup and wash the cells twice with PBS. Collect cells using rubber scraper or Trypsin. Collect to 15ml flask.

  4. Wash cells sequentially with 2.5ml cold PBS Buffer 1 and Buffer 2 (at this stage the nuclear pellet can be frozen in liquid nitrogen and stored at -80oc).

  5. Ressuspend cells at 0.5-1ml lysis buffer (add protease inhibitor 1:100), transfer to an eppendorf and sonicate. Sonication 6 cycles, each 6 min (30 ON, 30 OFF).

  6. Spin for 15 min at 13000 rpm in 4oC centrifuge and transfer sup to a new tube.

  7. Run a small sample (5μl) on agarose gel. The majority of DNA should run around the 1 Kb marker.

  8. Reverse the crosslink using the Chelex resin protocol (detailed below) of a small sample (20 µl for 10 plate) and measure the DNA concentration. (This will serve as your input control for the IP).


Chelex resin for input1 (if normalization of the samples before IP is needed)

  1. add 100μl of 10% Chelex (10 g/100 ml H2O)

  2. vortex samples

  3. Boil samples for 10 minutes at 95oC.

  4. allow the samples to cool to R.T

  5. Add 100 mg/ml Proteinase K (3μl of 20 μl/μg stock for a sample) and incubate for 30 min at 55oC while shaking.

  6. Boil samples for 10 minutes.

  7. Centrifuge samples and transfer supernatant to a new test tube

  8. Add 100μl of H2O to the beads, vortex, centrifuge and combine supernatant with the previous one. Centrifuge again to get rid of chelex beads.

  9. Check DNA concentration Dilute the different samples in lysis buffer so the concentration of all the samples is the same.


Immunoprecipitation

  1. Preparation of the samples for IP:

For 1 sample: 50μl of the lysate + 450μl of dilution buffer.
Calculation how much is needed for IP, and proceeding to preclear step.

  1. Preclear2 of the samples o/n at 4oC rotor with 10μl (nProtein A Sepharose, 4 Fast Flow) ChIP beads (for a sample), washed with dilution buffer.

At the same time:

  1. Beads + antibody - Bind the antibody to 15μl (nProtein A Sepharose, 4 Fast Flow) ChIP beads o/n at 4oC rotor, in dilution buffer. For example:

  1. only beads (no antibody)

  2. IgG 0.5μl

  3. Anti-Flag (for example) 2.5μl

  1. Centrifuge beads + antibody tubes and wash once with dilution buffer.

  2. Centrifuge preclear samples and transfer sup to the IP tubes (beads + antibody).
    Incubate o/n at 4oC rotor.

  3. Wash IP tubes once with 750μl TSE150, TSE500 and buffer 3, then twice with TE buffer. Each wash is 3min at R.T rotor.

Elution2

  1. For IP: to each washed tube with the IP beads, add 150μl of elution buffer.

For input: take 50μl input and add 150μl of elution buffer (in total 200μl).

  1. RNaseA treatment (stock 20 μg/μl) with a final concentration of 0.2 μg/μl.
    for IP: 1.5μl, for input: 2μl.

Incubate at 37oC bath for 1h, shaking every 15min.

  1. ProteinaseK treatment (stock 20 μg/μl) with a final concentration of 0.2 μg/μl.
    for IP: 1.5μl, for input: 2μl.

Incubate at 55oC dry bath for 1h, shaking every 15min.

Decrosslinking2

  1. After the incubations with the enzymes, centrifuge for 2min at 3500rpm, R.T.
    Transfer sup - the elution of the IP to new tubes.

  2. Incubate input and IP elution at 65oC dry bath for 4h, shaking every 30min.

DNA purification

Purify the DNA using PCR cleanup Kit.
MACHEREY-NAGEL kit – PCR clean-up protocol (5.1) suitable for DNA concentration.
*3 washes with NT3 buffer.

Elute the DNA with 85μl DDW.

Proceed to qPCR or ChIP-seq.







Buffers

1.25M Glycine pH 8.5

For 40ml: 3.752gr of Glycine + titer with HCl.


Wash buffer 1


Final concentration

To 50 ml

Stock concentration

HEPES pH 6.5

10mM

500 μl

1M

EDTA

10mM

1 ml

0.5M

EGTA

0.5mM

50 μl

0.5M

Triton X-100

0.25%

125 μl

100%

H2O


47.2 ml


Wash buffer 2


Final concentration

To 50 ml

Stock concentration

HEPES pH 6.5

10mM

500 μl

1M

EDTA

1mM

100 μl

0.5M

EGTA

0.5mM

50 μl

0.5M

NaCl

200mM

2.5 ml

4M

H2O


47.35 ml


Lysis buffer


Final concentration

To 50 ml

Stock concentration

Tris pH 8

50mM

2.5 ml

1M

EDTA

10mM

1 ml

0.5M

SDS

1%

2.5 ml

20%

Protease inhibitors

1/100

Add fresh


H2O


44 ml


Dilution buffer


Final concentration

To 50 ml

Stock concentration

Tris pH 8

20mM

1 ml

1M

EDTA

2mM

200 μl

0.5M

NaCl

150mM

1.875 ml

4M

Triton X-100

1.84% (2)

920 μl

100%

SDS (2)

0.2% or 0.1%

500 μl or 250 μl

20%

H2O


42.3 ml


TSE150, TSE500


Final concentration

To 50 ml

Stock concentration

Tris pH 8.1

20mM

1 ml

1M

EDTA

2mM

200 μl

0.5M

Triton X-100

1%

500 μl

100%

SDS

0.1 %

250 μl

20 %

NaCl

150mM/ 500mM

1.875ml/6.25ml

4M

H2O


42.05 ml/38.55ml




Buffer 3


Final concentration

To 50 ml

Stock concentration

Tris pH 8

10mM

500 μl

1M

LiCl

250mM

12.5 ml

1M

NP-40

1%

5ml

10%

Deoxycholate

1%

5ml

10%

EDTA

1mM

100 μl

0.5M

H2O


26.9 ml


TE


Final concentration

To 50 ml

Stock concentration

Tris pH 8

10mM

500 μl

1 M

EDTA

1mM

100 μl

0.5 M

H2O


49.4 ml


Elution buffer2


Final concentration

To 20 ml

Stock concentration

SDS

1%

1 ml

20%

NaHCO3

50mM

1 ml

1M

NaCl

140mM

700 μl

4M

H2O


17.3 ml




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