1
Formaldehyde treatment
1-a
formaaldehyde treatment according to the protocol of the immunoprecipitation
(ChIP) kit (Upstate Biotechnology).
1-b
resuspend the cell pellet into 200 l of SDS lysis buffer/protease inhibitor
1-c
transfer into microcentrifuge tube  on ice 10 min
1-d
sonication at output 4, duty 70 for 10 sec
1-e
cfg 15K x 10 min at 4°C
1-f
transfer the sup to 15ml tube
1-g
add 2 ml of the chip dilution buffer, store 1/2 of the sample at -20°C as “PreIP”
1-h
mix the rest half with 40 l of tRNA / BSA / proteinA-Sepharse beads (0.2 mg/ml
tRNA, 0.5 mg/ml BSA, 0.05% azide)  rotate at 4°C for 30 min
1-i
cfg at 7K x 1 min
2-a
2-b
2-c
2-d
2-e
2-f
2-g
2-h
2-i
2-j
2-k
2-l
2-m
2-n
acetyl-histone IP
transfer the sup to another microcentrifuge tube, add -acetyl H3 Ab (1 mg/ml) 
4°C O/N
add 60 l of tRNA/BSA/proteinA-agarose beads  rotate at 4°C >90min
cfg the histon IP/protein A at 7K x 1min  discard the sup
add 1 ml of low salt wash buffer rotate 4 min at RT  7K x 1 min  discard sup
add 1 ml of high salt wash buffer rotate 4 min at RT  7K x 1 min  discard sup
add 1 ml of LiCl wash buffer rotate 4 min at RT  7K x 1 min  discard sup
add 1 ml of 1xTE rotate 4 min at RT  7K x 1 min  discard sup
= 2-g
add 250 l of the elution buffer, vortex  rotate at RT > 15 min  7K x 1 min 
transfer the sup to a new microcentrifuge tube
again add 250 l of the elution buffer to the original tube, vortex  rotate at RT
>15min  cfg at 7K x 1min  take the sup and combine
add 5M NaCl 20 l to the histon IP sample (500 l) & 40 l to PreIP sample (1 ml)
vortex  65°C for 4 hr (mix by inversion several times)
per 500 l of sample, add 10 l of 0.5M EDTA, 20 l of 1M TrisHCl (6.5), 1 l of
20 mg/ml proteinase K  45°C for 1hr
Phenol/CHCl3 ext  CHCl3 ext  EtOH ppt with NaOAc/glycogen  cfg  rinse
with 70% EtOH  dissolve in 150 l DDW  store 50 l at -20°C for real-time
PCR & use the rest 100 l (upto 5 g)
RNase treatment
DNA/DDW
100 g/ml RNaseA
100 l
100 l
/ 200 l
3-a
mix
3-b
 37°C for 1 hr
Phenol/CHCl3 ext  CHCl3 ext  EtOH ppt/glycogen  cfg  rinse with 70%
EtOH  dissolve in 150 l DDW  store 78 l at -20°C, use 72 l for further study
4-a
Blunting
DNA/DDW
5xT4 pol buffer (Invitrogen)
50 mM DTT
2 mM dNTPmix
72 l
20 l
1 l
5 l
2
4-b
T4 DNA pol (Invitrogen)
2 l
/ 100 l
 11°C for 15 min
Phenol/CHCl3 ex  CHCl3 ext  EtOH ppt/glycogen  cfg  rinse with 70%
EtOH
Kinasion
5-a
5-b
6-a
6-b
DDW
24 l
10x kinasion buffer
3 l
T4 PNK
3 l
/ 30 l
 37°C for 30 min
Phenol/CHCl3 ext  CHCl3 ext  EtOH ppt/glycogen  cfg  rinse with 70%
EtOH
dissolve in
dissolve in
Rsa I digestion
DDW
10xNEB buffer 1
Rsa I
40 l
5 l
5 l
/ 50 l
 37°C for 2 hr
Phenol/CHCl3 ext  CHCl3 ext  EtOH ppt/glycogen  cfg  rinse with 70%
EtOH dissolve in 18 l DDW  preheat at 65°C for 3 min on ice
7-a
DDW
DNA
50 M TAG adaptor
10 x ligation buffer
T4 DNA Ligase
Ligation of TAG adaptor
tester
12 l
0.5 g (or 2l)
10 l
3 l
3 l
/ 30 l
driver
48 l
2 g (or 8 l)
40 l
12 l
12 l
/ 120 l
16°C for overnight
Amplification of IP genome
8-a
DDW
10xPCR buffer
25 mM dNTP mix
DMSO
ligation mix
100 M TAG primer
Taq DNA pol (Invitrogen, NOT HotStart)

8-c
79.3 l
10l
1.2 l
5 l
1.5 l
1 l
1.5 l
/ 100 l
72°C for 5 min

95°C for 3 min

43 cycles of 95°C x 1 min, 77°C x 3 min

72°C for 10 min

4°C soak
check PCR products (5 l) by gel phoresis (should be a smear of 0.3 kbp ~ 2 kbp)
phenol/CHCl3 extraction  CHCl3 extraction 
3
add
8-d
8-e
1/2 volume 8M NH4OAc
4 volume EtOH
 dry ice
15K x 10 min  70% EtOH rinse  dry
dissolve in 50 l TE & check the DNA amount by spectrophotometer
Subtraction
9
9-a
Adaptor digestion
tester
driver
DDW
x l
y l
amplicon
5 g
30 g
100xBSA
1 l
10x NEBuffer 4
10 l
10 l
Enzyme
XmaI 5 l
SmaI 5 l
/ 100 l x 4 tubes
/ 100 l x 18 tubes
37°C for 2 hr
25°C for 2 hr
9-b
add
XmaI 5 l
SmaI 5 l
37°C >2hr
25°C >2hr
9-c
Phenol/CHCl3 extraction  CHCl3 extraction  use sup for spin column
(remove 5 l each for electrophoresis at step 10-g)
10
Spin column
10-a
apply the digested solutions onto MicroSpin S-400 HR columns (Amershma) to
remove adaptors (according to the manufacturer’s protocols)
(take 5 l for electrophoresis to check whether adaptors are removed)
10-b
to the column-eluent
add
1/10 vol
3M NaOAc
2.5 vol
EtOH
 mix and place on dry ice  cfg 15K x 10 min  rinse with 70% EtOH  dry
 dissolve in 50 l TE & check DNA ammount by spectrophotometer
11
11-a
11-b
11-a’
Ligation of subtraction adaptor
for 1st round
DDW
tester DNA
50 1st subtraction adaptor
10 x ligase buffer
T4 DNA ligase
x l
0.5 g
10 l
3 l
3 l / 30 l
16°C for O/N
70 l
Add TE
 Phenol/CHCl3 extraction
mix driver DNA 40 g with tester DNA with 1st adaptor 100 l
for 2nd round
DDW
RDA DNA
50 2nd subtractin adaptor
10x ligase buffer
x l
200 ng
10 l
3 l
4
T4 DNA ligase
11-b’
11-c
11-d
11-e
11-f
12
*
12-a
12-b
13
13-a
13-b
13-c
3 l / 30 l
16°C for O/N
70 l
Add
TE
 Phenol/CHCl3 extraction
mix driver DNA 40 g with 1st subtracted DNA with 2nd subtraction adaptor 50 l
CHCl3 extraction  NaOAc/EtOH ppt  dry ice
cfg at 15K x 10 min  rinse with 70% EtOH  dry on heat block
dissolve in
3 x EE
2 l
 tapping  vortex  spin down
add 3 x EE
2 l
/ 4 l
 vortex  add oil
96°C for 10 min
transfer heated (96°C) 5M NaCl 1.0 l  incubate at 67°C for 20~24 hr
Amplification of tester-specific amplicon
prepare PCR mix
DDW
150.6 l
DMSO
20 l
10xPCR buffer
20 l
25mM dNTP mix2.4 l
Taq pol
3 l
/
196 l
transfer heated (67°C) 1M NaCl 45 l to the DNA mix  vortex 85°C for 3 min
transfer 5 l of the DNAmix/NaCl to the PCR mix tube72°C for 5 min  95°C
 add 4 l of heated 50 M 1st (or 2nd for the 2nd subtraction) subtraction primer
 95°C for 3 min
PCR 10 cycles of
95°C for 1 min, 77°C for 3 min
 77°C for 5 min
 4°C
Mung Bean (MB) nuclease digestion
Add 10x MB buffer
20 l
MB nuclease (NEB)
10 l
mix and incubate at 30°C for 30 min
Phenol/CHCl3 extraction  Phenol/CHCl3 extraction  CHCl3 extraction 
add
1 l
glycogen
1/2 vol 8M NH4OAc
4 vol
EtOH
 mix &b cfg 15K x 10 min  rinse with 70% EtOH dry
 dissolve in
TE
150.6 l
add
DMSO
20 l
10xPCR buffer
20 l
25 mM dNTPmix
2.4 l
Taq pol
3 l
/ 196 l
95°C for 3 min
 add 50 M 1st (or 2nd) subtraction primer 4 l95°C for 3 min
 20 cycles of PCR
5
13-d
13-e
95°C for 1 min
77°C for 3 min /
 72°C for 5 min
 4°C
Phenol/CHCl3 extraction  CHCl3 extractinn  NH4OAc/EtOH ppt  dissolve in
TE & check DNA amount
go to 2nd round of subtraction at STEP 9
or (after the 2nd subtraction) ligation to pBlueScript/XmaI (CIP treated)