Biology Reference
In-Depth Information
1. Aspirate medium from infected cell monolayers and add 5 mL
of pre-warmed serum-free medium per 100 mm dish.
2. Add 37 % formaldehyde directly to culture medium to a fi nal
concentration of 1 %. Incubate for 10 min at 37 °C.
3. Quench cross-linking by the addition glycine to a fi nal concen-
tration of 125 mM and incubate 5 min at room temperature.
3.2 Formaldehyde
Cross-Linking
1. Aspirate medium and wash cells twice using ice-cold PBS
solution.
2. Add 600
3.3 Cell Lysis
L of PBS to each plate, scrape the cells from the
dish, and transfer into a clean 1.5 mL microfuge tube.
3. Pellet the cells by centrifugation for 5 min at 1,000 ×
g
at 4 °C.
4. Resuspend the cell pellet in 600
μ
l of SDS lysis buffer contain-
ing protease inhibitors (1 mM PMSF, 1
μ
μ
g/mL aprotinin,
g/mL pepstatin A).
5. Incubate the resuspended cells 10 min on ice. [Note: add pro-
tease inhibitors to buffer just prior to use. PMSF has a half-life
of approximately 30 min in aqueous solution].
1
μ
1. To shear the chromatin, merge the microfuge tubes contain-
ing cell lysate in an ice-water bath, and sonicate with four
rounds of 30 s pulses with 2 min intervals, output control at
fi ve, duty cycle at 50 % (Branson Sonifi er 450).
2. Clear the cell lysate by centrifuging at 16,000 ×
g
for 10 min at
4 °C.
3. Collect supernatant, divide into 500
3.4 Sonication
( See Note 1 )
μ
L aliquots in microfuge
tubes, and store samples at −80 °C.
4. Remove 50
L of each sonicated sample, this sample is input
DNA. It is used to quantify the DNA concentration and as a
control in the qPCR:
(a) Reverse the cross-linking by adding 500
μ
g/mL protein-
ase K, incubate at 65 °C for 4 h (or overnight).
(b) Recover DNA by phenol/chloroform extraction and
ethanol precipitation. Wash DNA with 70 % ethanol, air
dry.
(c) Suspend DNA pellet in 50
μ
L of TE. Read Absorbance at
260 nm of a 1:20 dilution of the sample.
(d) Run 2
μ
g of DNA on a 1 % agarose gel in comparison to
molecular weight DNA standards to assess chromatin
shearing effi ciency.
μ
3.5 Normalization
of Input Chromatin
by qPCR
qPCR gives accurate quantifi cation of target DNA, which allows
one to adjust and standardize the input DNA concentration for
each ChIP sample. Take 2
μ
L input DNA prepared in last step (
see
