Revision as of 14:49, 30 March 2014 by >Noi
DNA methylation assay - MONOD Project (BSPP capture)
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Sample list
- - Cancer cell lines (5 samples)
- - Differentiated H1 & H9 (BMP4 treatment, I also included H1 control, 3 samples)
- - Blood samples (3 samples)
Sample IDs tracking
Sample ID
|
Original ID
|
CANC_1 |
BXPC3
|
CANC_2 |
U87MG
|
CANC_3 |
PANC1
|
CANC_4 |
BE(2)C
|
CANC_5 |
T98G
|
SR-3 |
H9 p43 BMP4
|
MA-16 |
H1 p47 Control
|
MA-17 |
H1 p47 BMP-4
|
F10 |
UCLA-B-F10
|
G10 |
UCLA-B-G10
|
G11 |
UCLA-B-G11
|
Bisulfite conversion
- I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo and followed the protocol of the kit
Bisulfite conversion set up
Samples
|
Conc. (ng/ul)
|
Volume for 600ng (ul)
|
Go to tube
|
H2O
|
Lightning Conv Reagent
|
CANC_1 |
47.70 |
12.58 |
MN#1 |
7.42 |
130.00
|
CANC_2 |
45.40 |
13.22 |
MN#2 |
6.78 |
130.00
|
CANC_3 |
41.40 |
14.49 |
MN#3 |
5.51 |
130.00
|
CANC_4 |
47.10 |
12.74 |
MN#4 |
7.26 |
130.00
|
CANC_5 |
68.20 |
8.80 |
MN#5 |
11.20 |
130.00
|
SR-3 |
62.20 |
9.65 |
MN#6 |
10.35 |
130.00
|
MA-16 |
85.00 |
7.06 |
MN#7 |
12.94 |
130.00
|
MA-17 |
49.40 |
12.15 |
MN#8 |
7.85 |
130.00
|
F10 |
106.00 |
5.66 |
MN#9 |
14.34 |
130.00
|
G10 |
108.00 |
5.56 |
MN#10 |
14.44 |
130.00
|
G11 |
82.60 |
7.26 |
MN#11 |
12.74 |
130.00
|
NTC |
0 |
0 |
MN#12 |
20.00 |
130.00
|
Program
1. 98°C for 8 minutes
2. 54°C for 60 minutes
3. 4°C hold
- I elute with Elution buffer 16ul
- I used 1ul for ssDNA Qubit assay. The volume left after assay ~13ul.
Qubit ss DNA assay
- Dilute 200X (add 1ul of bis-cvt DNA in total 200 assay volume
Sample
|
Conc. in the Qubit
|
'
|
uL used
|
Dilution
|
Sample conc.
|
'
|
Yields in 13ul (ng)
|
% recovery
|
MN_1 |
175 |
ng/mL |
1 |
200 |
34.9 |
ng/ul |
453.7 |
75.62
|
MN_2 |
122 |
ng/mL |
1 |
200 |
24.5 |
ug/mL |
318.5 |
53.08
|
MN_3 |
122 |
ng/mL |
1 |
200 |
24.5 |
ug/mL |
318.5 |
53.08
|
MN_4 |
87.6 |
ng/mL |
1 |
200 |
17.5 |
ug/mL |
227.5 |
37.92
|
MN_5 |
108 |
ng/mL |
1 |
200 |
21.7 |
ug/mL |
282.1 |
47.02
|
MN_6 |
131 |
ng/mL |
1 |
200 |
26.2 |
ug/mL |
340.6 |
56.77
|
MN_7 |
107 |
ng/mL |
1 |
200 |
21.4 |
ug/mL |
278.2 |
46.37
|
MN_8 |
71.4 |
ng/mL |
1 |
200 |
14.3 |
ug/mL |
185.9 |
30.98
|
MN_9 |
61.5 |
ng/mL |
1 |
200 |
12.3 |
ug/mL |
159.9 |
26.65
|
MN_10 |
173 |
ng/mL |
1 |
200 |
34.6 |
ug/mL |
449.8 |
74.97
|
MN_11 |
72.9 |
ng/mL |
1 |
200 |
14.6 |
ug/mL |
189.8 |
31.63
|
BSPP capture set up
Probe calculation
Probe:taeget
|
200:1
|
Unit
|
Calculation
|
Probe size (number of individual probe in oligo mix) |
83447 |
probes |
|
Human bis-cvt gDNA required |
200 |
ng |
|
Human gDNA MW |
1.822E+12 |
g/mole |
3.0E+09 * 607.4Da/bp
|
Human gDNA( 200ng) |
1.098E-19 |
mole |
200E-09g / 1.822E+12g/mole
|
Probe required (200:1) |
2.195E-17 |
mole |
200 * 1.098E-19mole
|
Probe mix MW (size 83447probes, 110nt) |
2.788E+09 |
g/mole |
83447 probes * 110bp *303.7Da/bp
|
Mass of probe required |
6.119E-08 |
g |
2.195E-17mole * 2.788E+09g/mole
|
Mass of probe required |
61.20 |
ng |
|
Probe conc |
11.9 |
ng/ul |
|
Volume of probe |
5.14 |
ul |
|
Probe & AmpLigase Buffer mix
Components
|
Volume (ul)
|
12.5x
|
10X AmpLigase Buffer |
2.00 |
25.00
|
MONOD (11.90ng/ul) |
5.14 |
64.25
|
Toal |
7.14 |
|
Bis-cvt samples
|
Conc. (ng/ul)
|
Volume for 200ng (ul)
|
Bis-cvt samples
|
H2O (ul)
|
MONOD probe (61.20ng)
|
10X AmpLigase Buffer
|
Total (ul)
|
Converted DNA amount (ng)
|
MN_1 |
34.90 |
5.73 |
MN_1 |
7.13 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_2 |
24.50 |
8.16 |
MN_2 |
4.70 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_3 |
24.50 |
8.16 |
MN_3 |
4.70 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_4 |
17.50 |
11.43 |
MN_4 |
1.43 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_5 |
21.70 |
9.22 |
MN_5 |
3.64 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_6 |
26.20 |
7.63 |
MN_6 |
5.23 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_7 |
21.40 |
9.35 |
MN_7 |
3.51 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_8 |
14.30 |
13.00 |
MN_8 |
0.00 |
5.14 |
2.00 |
20.14 |
185.90
|
MN_9 |
12.30 |
13.00 |
MN_9 |
0.00 |
5.14 |
2.00 |
20.14 |
159.90
|
MN_10 |
34.60 |
5.78 |
MN_10 |
7.08 |
5.14 |
2.00 |
20.00 |
200.00
|
MN_11 |
14.60 |
13.00 |
MN_11 |
0.00 |
5.14 |
2.00 |
20.14 |
189.80
|
MN_12 |
0.00 |
0.00 |
MN_12 |
12.86 |
5.14 |
2.00 |
20.00 |
0.00
|
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h
-> add 2.5ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
- Note: Concentration of Hemo Klentaq was not provided, so I assumeed it has the same concentration as Stoffel Fragment
SLN mix solution
Components
|
Stock conc.
|
Unit
|
Final conc.
|
Unit
|
Prepare volume (ul) total 40ul
|
Hemo Klentaq |
10 |
U/ul |
2 |
U/ul |
8.00
|
AmpLigase |
5 |
U/ul |
0.5 |
U/ul |
4.00
|
dNTP |
1 |
mM |
100 |
uM |
4.00
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
4.00
|
H2O |
|
|
|
|
20.00
|
2014-03-05
PCR Amplification (TEST)
Components
|
1x rxn
|
12.5x rxn mix
|
Captured template |
4.00 |
0.00
|
10uM AmpF6.4Sol |
0.50 |
6.25
|
10uM AmpR6.3Indx1 |
0.50 |
6.25
|
2X KAPA SYBR MM |
12.50 |
156.25
|
H2O |
7.50 |
93.75
|
Total |
25.00 |
|
- Aliquot 21ul, add 4ul of circularized DNA
Program (Eppendorf Realplex)
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
qPCR curves
File:2014-03-05 qPCR TEST MONOD capture.png
Verified amplified products in 6% TBE gel
- Loaded 3ul of PCR product in 6% TBE gel
File:ZhangLab 2 2014-03-05 13hr 23min TEST PCR MONOD MN 1-8.jpg File:ZhangLab 2 2014-03-05 13hr 23min TEST PCR MONOD MN 9-11.jpg
- If everything works well, I will set up duplicate reactions of 50ul with 20% (v/v) of template
Components
|
1x rxn
|
25x rxn mix
|
Captured template |
10.00 |
0.00
|
10uM AmpF6.4Sol |
1.00 |
25.00
|
10uM AmpR6.3IndX(X=81-92) |
1.00 |
0.00
|
2X KAPA SYBR MM |
25.00 |
625.00
|
H2O |
13.00 |
325.00
|
Total |
50.00 |
|
- Aliquot 39ul, add 1ul of 10uM AmpR6.3IndX, add 10ul of circularized DNA
Program (Eppendorf Realplex)
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
qPCR curves
I took the average of the replicates
File:2014-03-05 qPCR MONOD capture.png
- - Stopped MN_1-8 at 14 cycles
- - Stopped MN_9-11 at 18 cycles
- - Pool and purify PCR product with 0.7 volume of AMPure beads
- - Eluted with 65ul H2O
- - Quantify concentration in 6% TBE gel (load 1.5ul of purified libraries)
- File:ZhangLab 2 2014-03-05 17hr 20min MONOD SeqLib PQind.jpg
Sample IDs
|
Original IDs
|
Indexes
|
Conc. (ng/ul)
|
Total amount in 65ul (ng)
|
Volume for 50ng (ul)
|
MN#1 |
BXPC3 |
Ind81 |
2.16 |
140.17 |
23.19
|
MN#2 |
U87MG |
Ind82 |
3.14 |
204.13 |
15.92
|
MN#3 |
PANC1 |
Ind83 |
6.28 |
408.34 |
7.96
|
MN#4 |
BE(2)C |
Ind84 |
3.76 |
244.61 |
13.29
|
MN#5 |
T98G |
Ind85 |
1.90 |
123.48 |
26.32
|
MN#6 |
H9 p43 BMP4 |
Ind86 |
8.26 |
536.74 |
6.06
|
MN#7 |
H1 p47 Control |
Ind87 |
2.77 |
180.10 |
18.05
|
MN#8 |
H1 p47 BMP-4 |
Ind88 |
1.08 |
70.34 |
46.20
|
MN#9 |
UCLA-B-F10 |
Ind89 |
3.32 |
216.00 |
15.05
|
MN#10 |
UCLA-B-G10 |
Ind90 |
5.65 |
367.18 |
8.85
|
MN#11 |
UCLA-B-G11 |
Ind91 |
1.50 |
97.31 |
33.40
|
- - Pool equal amount of sequencing libraries and performed PAGE size-selection in 2 of 5-well 6% TBE gels at the band ~375bp
- File:ZhangLab 2 2014-03-05 18hr 59min PAGE-SS.jpg
- - Elute with 24ul H2O.
- - Verify sequencing libraries in 6% TBE gel by loading 2ul
- File:ZhangLab 2 2014-03-06 18hr 21min verify MONOD-SeqLib.jpg
- -Hand to Alan for concentration quantification by qPCR
Sequencing ibrary IDs: NP-BSPP-MONOD_81-91_Mar2, 2014
Sequencing run: Dr. Zhang suggested to do MiSeq PE run, 150+7+150 bp
- - MiSeq run: MONOD_81_91 on March 3rd, 2014