Noi/NOTES/2014-3-2

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DNA methylation assay - MONOD Project (BSPP capture)

[Link to calendar]
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