Noi/NOTES/2014-3-2

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DNA methylation assay - MONOD Project (BSPP capture)[edit]

[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[edit]

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[edit]

  • I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo and followed the protocol of the kit

Bisulfite conversion set up[edit]

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[edit]

  • 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[edit]

Probe calculation[edit]

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[edit]

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 assumed it has the same concentration as Stoffel Fragment

SLN mix solution[edit]

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[edit]

PCR Amplification (TEST)[edit]

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[edit]

File:2014-03-05 qPCR TEST MONOD capture.png

Verified amplified products in 6% TBE gel[edit]

  • 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[edit]

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