Chromatin Immunoprecipitation Assay
J. Kim Kemper Lab (for HepG2 cell system)
*15cm plate  3~4 IP
<Ligand Treatment>
Treat ligand to HepG2 cells with serum-free media
<Formaldehyde cross-link>
1.
Take 2ml of 37% formaldehyde solution (by 5ml glass tube) and put
into boiling water bath for 10sec  set aside @RT
 Take different volume of formaldehyde depending on how many plates
you have.
2.
Wash the cells with warm(@RT) PBS and cross link with 1%
formaldehyde in 20ml PBS for 10~15min @RT by gently shaking.
(540ul of 37% formaldehyde in 20ml PBS)
3.
Quench with 2M glycine @RT for 3min (1.25ml of 2M glycine to 20ml)
4.
Wash cells with ice-cold PBS(DTT, PMSF) and collect cells with 10ml
of DTT solution (100mM Tris pH9.5, 10mM DTT)
5.
Incubate @RT for 10min and lysate cells by vortexing every 3min
6.
Spin @5000rpm, 4C for 3min. Remove supernatant
<Sonication & Preclearing>
7.
Resuspend cell pellet with 700ul of SDS-lysis buffer (2mM EDTA,
50mM Tris pH8.0, final concentration of SDS should be 1%)  divide
into 2 tubes (each contains ~400ul)
8.
Sonicate with 80% output for 30 sec until solution is going to be clear
(10 sec X 3)
 Modify by yourself how long sonication should be carried out.
9.
Spin @13000rpm, 4C for 10min
10.
Collect supernatant and add 2-fold volume of dilution buffer (20mM
TrisHCl pH8.0, 2mM EDTA, 200mM NaCl, 1% Triton X-100, 0.1% Nadeoxycholate)
11.
Add (40ul Normal Goat Serum+100ul of 25% Protein A/G sepharose+
5ug rabbit IgG) per 15cm plate.
12.
Incubate @4C for 30min~1hour.
<Immunoprecipitation >
13.
Spin @8000rpm, 4C for 1min. Take supernatant only and combine all
of them.
14.
Save 1/10 of IP volume for total chromatin.
15.
Divide supernatant into IP tubes and add optimized amount of Ab 
O/N incubation @4C or at least more than 4hour incubation @4C.
<Collect Immune complex>
16.
Collect immune complex by adding 40ul of 25% protein A/G sepharose
 rotate @4C for 1~2hrs, no more than 2hours.
<Wash and Elute>
17.
Spin @8000rpm, 4C for 1min. Remove supernatant only
18.
Wash protein A/G sepharose with 500ul of TSE I once (+DTT, PI)
 rotate @4C for 5min
 centrifuge @8000rpm for 1min, remove supernatant.
19.
Wash with 500ul of TSE II once (+DTT, PI)
 rotate @4C for 5min
 centrifuge @8000rpm for 1min, remove supernatant.
20.
Wash with 500ul of TSE III once (+DTT, PI)
 rotate @4C for 5min
 centrifuge @8000rpm for 1min, remove supernatant.
21.
Wash with 500ul of TE twice
 rotate @4C for 5min
 centrifuge @8000rpm for 1min, remove supernatant.
22.
Remove all supernatant as you can, and add 410ul~420ul of elution
buffer (0.1M NaHCO3, 1%SDS)  Don’t forget Total. Add elution
buffer to total tube to make the volume as 400ul.
 Rotate @RT for 20min ~1hour
<Reverse Cross-linking>
23.
After elution, spin @8000rpm for 1min.
 Transfer only 400ul of supernatant to new tubes
24.
Add NaCl to every tube. Final NaCl concentration is 200mM.
 Add 16ul of 5M NaCl to tube.
 Incubate @65C for at least 6hrs or O/N.
<Purification of DNA>.
25.
Add 8ul of 0.5M EDTA, 16ul of 1M TrisHCl pH 6.5 to every tube.
 At the same time, add 1.5ul of 25mM Proteinase K to every tube.
 Incubate @65C for additional 30min~1hour
26.
Chloroform extraction
 Add 400ul of CHCl3 to every tube
 Vortex very well and spin @13000rpm for 5min
27.
Meanwhile, label new tubes and add 1ul of yeast tRNA, 35ul of
NaOAc(pH7.0) to each tube.
28.
Transfer upper layer about 350ul to new tubes and add 870ul of 100%
EtOH.
 Vortex very well and store @-20C for at least >2hrs or O/N.
<PCR>
29.
Spin @13000rpm, 4C for 30min
 Remove supernatant and air-dry for 10min.
30.
Dissolve DNA pellet with 50ul of TE buffer or DNase free water
 For total, add 100ul of TE or water.
 Vortex very well to resuspend pellet completely.
31.
PCR step
DNA sample
: 5ul
(total : 2ul )
10X PCR bf
: 5ul
50mM MgCl2
: 3.5ul (modify as your desire)
25mM dNTP
: 0.35ul
100uM Primer F : 0.1ul
100uM Primer B : 0.1ul
Taq polymerase : 0.2ul
PCR H2O
: 35.7ul
50ul /rxn