Good thing takes time

ChIP (Lyu, YL et. al. MCB 2006).

Whole brains of TOP2b (pool of two Top2b+/+ and three top2b+/2

brains) and top2b2/2 (pool of five brains) embryos were dissected at E18.5 in

Dulbecco’s modified Eagle’s medium (DMEM) (with 10% fetal bovine serum

[FBS]). Brain tissues were minced and dislodged to single-cell suspensions in

DMEM–10% FBS using an Eppendorf pipette tip. Cells were then filtered

through a 100-mm cell strainer. Neutral-buffered formaldehyde was added to a

final concentration of 1%, and cells were fixed for 15 min at 4°C. Glycine was

then added to a final concentration of 0.125 M to stop the cross-linking reaction,

and cells were successively washed in wash buffer 1 (0.25% Triton X-100, 10 mM

EDTA, 0.5 mM EGTA, 10 mM Tris-HCl, pH 8.0), and wash buffer 2 (0.2 M

NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl, pH 8.0) and then

resuspended in resuspension buffer (1 mM EDTA, 0.5 mM EGTA, 10 mM

Tris-HCl, pH 8.0). Chromatin was sheared by sonication to fragments of _4 kb.

Chromatin solution was then adjusted to 1X RIPA (50 mM HEPES, pH 7.5, 1%

Triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS],

140 mM NaCl, protease inhibitor cocktail [Roche]). Immunoprecipitation (IP)

was performed using anti-TopII b antibody (Santa Cruz) at 4°C overnight. No

antibody was added for the control samples. Protein A-agarose beads were then

added to either II b IP mixtures or control mixtures to capture IP complexes or

serve as controls. The agarose beads were successively washed (twice for each

wash) in ChIP lysis buffer (50 mM HEPES, pH 7.5, 140 mM NaCl, 1% Triton

X-100, 0.1% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail), highsalt

ChIP lysis buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 1% Triton X-100,

0.1% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail), ChIP wash

buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium

deoxycholate, 1 mM EDTA), and TE (10 mM Tris-HCl, pH 8.0, and 1 mM

EDTA). The IP complex was then eluted twice with 75 _l of chromatin elution

buffer (50 mM Tris-HCl, pH 8.0, 1% SDS, 10 mM EDTA) at 65°C for 10 min.

Supernatants were combined and incubated at 65°C overnight to reverse protein-

DNA cross-links. For the input control, 1/100 of the chromatin solution taken for

IP was added to 150 ml of elution buffer and incubated at 65°C overnight. After

reversal, DNA was purified using the PCR purification kit (QIAGEN). DNA was

eluted with 100 ml elution buffer (10 mM Tris-HCl, pH 8.0). For PCR amplifi-

cation, 1 ml of the elution was used in each reaction mixture. The PCR primers

(listed below) were designed to amplify 200 bp of a DNA sequence located

within a 500-bp region upstream of the transcription start site for each gene of

interest.

ChIP stock solution I: 10X

Used for: wash buffer 1, wash buffer 2, ChIP wash buffer, Chromatin elution buffer

10 mM EDTA

100 mM Tris pH8.0

5mM EDTA

ChIP stock Solution II: 10X

Used for: RIPA, ChIP lysis buffer, high salt ChIP lysis buffer

500 mM Hepes pH 7.5

10% Triton X100

1% SDS

1% Sodium deoxycholate

Protease inhibitor (Roche)

Difference with Upsate protocol

1. add formaldehyde directly to cell medium
2. use SDS lysis buffer to cell before sonication
3. reverse DNA by adding NaCl at 65oC for 4 hours