giles 23 april 09
TRANSCRIPT
Following to be done for ArdC, ProP and AltB at
mid and late G2
◦ Quantification of transcription
◦ Quantification of PolII occupancy
◦ Quantification of H3/H4 replacement
Perform high resolution mapping at mid and late
G2 for AltB
Showed change in
AltB expression as
expected
Showed possible
enrichment of PolII on
AltB in late G2
0
1000
2000
3000
4000
5000
6000
ArdC ProP AltB
Mid G2
Late G2
-20
0
20
40
60
80
100
120
140
ArdC ProP AltB
Mid G2
Late G2
Late G2 Mid G2
Imp Un IP Imp Un IP
Following to be done for ArdC, ProP and AltB at
mid and late G2
◦ Quantification of transcription
◦ Quantification of PolII occupancy
◦ Quantification of H3/H4 replacement
Perform high resolution mapping at mid and late
G2 for AltB
-230 -156
Comparison of mononucleosomes from control and flag incorporated cells
-8
-6
-4
-2
0
2
4
6
8
10
1 2 3 4 5 6 6 7
8a
8b 9
10
11a
11B
12
13
14
15
16
17
18
19
20
Ard
C
Pro
P
DC
t
MC-C MF-F
Determine relative amplification from DNA cut 5 min and 15 min compared to uncut
0
5
10
15
20
25
1 2 3 4 5 6 7 8A 8B 9 10 11A 11B 12 13 14 15 17 19 ArdC
dCt5 dCt15
-4
-3
-2
-1
0
1
2
3
4
1 2 3 4 5 6 7 8a 8b 9 10 11a 11b 12 13 14 15 16 17 18 19 20
Mid G2 Late G2
100bp
-230 -156
Primer extension assay
-230 -156
Tried to quantify/amplify of different products in primer extension reaction
-230 -156
Mid G2 Late G2
PE Q1 Q2 Q3 Q4 Q5 PE Q1 Q2 Q3 Q4 Q5
P1/P2 P1 P2P1/P2 P1 P2 P1/P2 P1 P2P1/P2 P1 P2
Neat 1:100 Neat 1:100
Mid G2 Late G2
-230 -156
Use reconstituted 5s nucleosomes
Confirm position ◦ restriction digest
◦ New method
Apply new method to map completely AltB