Biology Reference
In-Depth Information
Subheading
3.4
,
step 4
) as an template to measure viral genome
enrichment by qPCR. Go to Subheading
3.8
. for details of qPCR
protocol.
3.6 Immuno-
precipitation
and Reversal
of Cross-Linking
All following manipulations are performed at 4 °C and all wash
buffers contain protease inhibitors.
1. Aliquot predetermined volume of chromatin (100
g total
DNA) to 15 mL conical tubes and dilute cell superant tenfold
in IP Dilution Buffer containing protease inhibitors (
see
above).
2. Add 80
μ
L of salmon sperm DNA-protein A agarose slurry to
preclear samples. Incubate for 1 h at 4 °C with gentle rotation.
(Protein A agarose is supplied as 50 % slurry in ethanol-
containing buffer, it should be washed with IP dilution buffer
prior to use).
3. Spin samples at 100 ×
g
for 1 min at 4 °C. Transfer superna-
tants to new tubes with 15
μ
g
of monoclonal antibody (
see
Note 2
). Incubate at 4 °C over-
night with rotation.
4. On the next day, add 60
μ
L of polyclonal antibody or 2
μ
L of pre-washed salmon sperm
DNA-protein A agarose and incubate at 4 °C for 1 h with
rotation.
5. Pellet the beads at 100 ×
g
for 1 min.
6. Carefully aspirate supernatant without disturbing beads.
7. Resuspend beads in 1 mL low salt wash buffer and transfer
into a clean 1.5 mL microfuge tube. Incubate the samples at
4 °C for 10 min with rotation, followed by brief centrifugation
to pellet beads.
8. Carefully aspire supernant.
9. Using this technique, perform the following additional washes
of the samples: once with 1 mL of high salt wash buffer, once
with 1 mL of LiCl wash buffer, and twice with 1 mL of TE
buffer (
see
Note 3
).
10. Add 150
μ
L of freshly prepare elution buffer to elute immune
complex. Incubate at room temperature for 15 min with
rotation.
11. Centrifuge samples at 1,500 ×
g
for 1 min and transfer super-
natant to another clean microfuge tube.
12. Repeat elution and combine the two elution steps supernatant
in the same tube (~300
μ
L in total).
13. Reverse formaldehyde cross-linking by adding 12
μ
μ
L 5 M
NaCl, 6
μ
L 0.5 M EDTA, 12
μ
L 1 M Tris-HCl, pH 6.5, and
7.5
L 20 mg/mL proteinase K and incubate at 65 °C for 4 h
(or overnight).
μ
