DAPI
description
Transcript of DAPI
B
DAPI DNA merge
S
P
A
CENP-A
HDAC1
Aurora B
C
Centrifugation on 15% sucrose 200 g, 10 min
Centrifugation on 30% sucrose 2500 g, 20 min
Centrifugation on 30% sucrose 27000 g, 30 min
Elimination of unbroken nuclei
Elimination of nucleoli
Supernatant (S) = low density fraction
Pellet (P) = chromocenters
Nuclei + 2 mM TEA, 0.2 mM MgCl2, 0.2% TritonSonication
Isolation of the chromocenters
200400
100
bp
S P
- + - +
minsat
E
Sir2
Survivin
S P
Ferri et al. Supplemental Figure 1
S P
minsat
200100
400600bp
15
40
15
60
120
kDa
D
minsat
rDNA
H3A
c
H3K
9Me3
CE
NP
-A
bea
ds
INP
UT
IP SUP
H3A
c
H3K
9Me3
CE
NP
-A
bea
ds
Tot
al D
NA
Mar
ker
100b
p
majsat
Ferri et al. Supplemental Figure 2
300500
100
300500
100
bp
300100
200300
100
400600
15002000
bp
- +- +AS NOC
min
sat
-actin
AS
0 400
0
100
200
200 600
G0/G1: 33%S: 57%
G2/M: 10%NOC
0 400
0
100
200
200 600
G0/G1: 2%S: 12%
G2/M: 86%
Ferri et al. Supplemental Figure 3
PI
coun
ts
PI
coun
ts 200
300400500
bp
1: RNA 2’OMe (1-27) 5’biot-GGAAACAUGAUAAAAACCACAGUGUAG-3’
2: DNA (1-27) 5’biot-GGAAACATGATAAAAACCACAGTGTAG-3’
3: DNA/lna (4-27) 5’-aACaTGaTAaAAaCCaCAgTGtAG-3’biot
4: DNA/lna (89-115) 5’-tGTgTAtATcAAtGAgTTaCAaTG-3’biot
5: DNA/lna (4-27) 5’biot-aACaTGaTAaAAaCCaCAgTGtAG-3’
murine minor satellites 120 nt repeat unit consensus sequence :
5’GGAAACATGATAAAAACCACAGTGTAGAACATATTAGATGAGTGAGTTACACTGAAA
AACACATTCGTTGGAAACGGGATTTGTAGAACAGTGTATATCAATGAGTTACAATGAG
AAACAT-3’
Ferri et al. Supplemental Figure 4
inpu
t
1 2 3 4 5
RNA minsat
MEL nuclear extractsStreptavidin
beads
Biotinylated oligonucleotide
In vitro transcribed RNA