Noi/NOTES/2014-3-30

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DNA methylation assay - MONOD Project, MONOD V3 12k probe set (BSPP capture)[edit]

[Link to calendar]
Sample list

Sample list will be the same as experiment in 2013-03-02
- Cancer cell lines (5 samples)
- Differentiated H1 & H9 (BMP4 treatment, I also included H1 control, 3 samples)
- Blood samples (3 samples)
  • I wanted to compare Hemo KlenTaq and Stoffel Fragment, so I will include one of cancer sample with Stoffel Fragment.

Probe preparation[edit]

[[1]]

Sample IDs tracking[edit]

Bisulfite conversion[edit]

  • * I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo (lot Mar 2014) 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 Total (ul) Samples
CANC_1 47.70 12.58 MN_1 7.42 130.00 150.00 CANC_1
CANC_2 45.40 13.22 MN_2 6.78 130.00 150.00 CANC_2
CANC_3 41.40 14.49 MN_3 5.51 130.00 150.00 CANC_3
CANC_4 47.10 12.74 MN_4 7.26 130.00 150.00 CANC_4
CANC_5 68.20 8.80 MN_5 11.20 130.00 150.00 CANC_5
SR-3 62.20 9.65 MN_6 10.35 130.00 150.00 SR-3
MA-16 85.00 7.06 MN_7 12.94 130.00 150.00 MA-16
MA-17 49.40 12.15 MN_8 7.85 130.00 150.00 MA-17
F10 106.00 5.66 MN_9 14.34 130.00 150.00 F10
G10 108.00 5.56 MN_10 14.44 130.00 150.00 G10
H11 102.00 5.88 MN_11 14.12 130.00 150.00 G11
NTC_HemoKlenTaq 0 0 MN_12 20.00 130.00 150.00 NTC
CANC_1 47.70 12.58 MN_13 7.42 130.00 150.00 CANC_1
NTC_Stoffel 0 0 MN_14 20.00 130.00 150.00 NTC
- Sample G11 was not enough, so I changed to smaple H11.
Program 

1. 98°C for 8 minutes
2. 54°C for 60 minutes
3. 4°C hold
  • I elute with Elution buffer 15ul
  • 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 Unit uL used Dilution Sample conc. (ng/ul) Yields in 13ul (ng) % recovery
MN_1 159 ng/mL 1 200 31.90 414.70 69.12
MN_2 145 ng/mL 1 200 29.10 378.30 63.05
MN_3 113 ng/mL 1 200 22.50 292.50 48.75
MN_4 107 ng/mL 1 200 21.40 278.20 46.37
MN_5 116 ng/mL 1 200 23.10 300.30 50.05
MN_6 138 ng/mL 1 200 27.60 358.80 59.80
MN_7 126 ng/mL 1 200 25.10 326.30 54.38
MN_8 125 ng/mL 1 200 25.00 325.00 54.17
MN_9 101 ng/mL 1 200 20.20 262.60 43.77
MN_10 252 ng/mL 1 200 50.50 656.50 109.42*
MN_11 141 ng/mL 1 200 28.10 365.30 60.88
MN_13 160 ng/mL 1 200 32.00 416.00 69.33
- * This sample always has too high recovery rate. This is consistent with previous experiment. I will use double amount for capture since there might be some contaminant causing the background.

BSPP capture set up[edit]

Probe calculation[edit]

MONOD_V3_12k, size 12,000

Probe:taeget 500:1 Unit Calculation
Probe size (number of individual probe in oligo mix) 12,000 probes
Human bis-cvt gDNA required 250 ng
Human gDNA MW 1.822E+12 g/mole 3.0E+09 * 607.4Da/bp
Human gDNA( 200ng) 1.372E-19 mole 250E-09g / 1.822E+12g/mole
Probe required (500:1) 6.860E-17 mole 500 * 1.098E-19mole
Probe mix MW (size12,000probes, 110nt) 4.018E+08 g/mole 12,000 probes * (110bp *303.7Da/bp+79Da)
Mass of probe required 2.757E-08 g 6.860E-17mole * 4.018E+08g/mole
Mass of probe required 27.57 ng
Probe conc 12.2 ng/ul
Volume of probe 2.26 ul
- I increased bis-DNA to 250ng.

Probe & AmpLigase Buffer mix[edit]

Components Volume (ul) 14.5x
10X AmpLigase Buffer 2.00 29.00
MONOD_V3_12k (12.2 ng/ul) 2.26 32.77
Toal 4.26
Bis-cvt samples Conc. (ng/ul) Volume for 250ng (ul) Bis-cvt samples H2O (ul) MONOD probe (61.20ng) 10X AmpLigase Buffer Total (ul) Converted DNA amount (ng)
MN_1 31.90 7.84 MN_1 7.90 2.26 2.00 20.00 250.00
MN_2 29.10 8.59 MN_2 7.15 2.26 2.00 20.00 250.00
MN_3 22.50 11.11 MN_3 4.63 2.26 2.00 20.00 250.00
MN_4 21.40 11.68 MN_4 4.06 2.26 2.00 20.00 250.00
MN_5 23.10 10.82 MN_5 4.92 2.26 2.00 20.00 250.00
MN_6 27.60 9.06 MN_6 6.68 2.26 2.00 20.00 250.00
MN_7 25.10 9.96 MN_7 5.78 2.26 2.00 20.00 250.00
MN_8 25.00 10.00 MN_8 5.74 2.26 2.00 20.00 250.00
MN_9 20.20 12.38 MN_9 3.36 2.26 2.00 20.00 250.00
MN_10 50.50 9.90 MN_10 5.84 2.26 2.00 20.00 499.95**
MN_11 28.10 8.90 MN_11 6.84 2.26 2.00 20.00 250.00
MN_13 32.00 7.81 MN_13 7.93 2.26 2.00 20.00 250.00
NTC_HemoKlentaq 0.00 0.00 NTC 15.74 2.26 2.00 20.00 0.00
NTC_Stoffel 0.00 0.00 NTC_Stoffel 15.74 2.26 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; 200uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

SLN mix solution (Hemo KlenTaq)[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume (ul) total 125ul
Hemo Klentaq 10 U/ul 2 U/ul 25.00
AmpLigase 5 U/ul 0.5 U/ul 12.50
dNTP 10 mM 200 uM 2.50
10x AmpLigase Buffer 10 x 1 x 12.50
H2O 72.50
- I prepare extra for MONOD_GP1_V4 capture experiment

SLN mix solution (Stoffel Fragment)[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume (ul) total 10ul
Stoffel Fragment 10 U/ul 2 U/ul 2.00
AmpLigase 5 U/ul 0.5 U/ul 1.00
dNTP 1 mM 200 uM 2.00
10x AmpLigase Buffer 10 x 1 x 1.00
H2O 4.00

Amplification[edit]

- Add 20% v/v of captured reaction in 100ul PCR (KAPA)
Program (Eppendorf Realplex)
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
- Sample MN13 stopped at 20 cycles
- Sample MN1,3, 6 stopped at 21 cycles
- Samples MN2,4,5,7-11 stopped at 23 cycles
- Verify 4ul of PCR product in 6% TBE gel
File:ZhangLab 2 2014-04-02 13hr 46min MONOD v3capture gel1.jpg File:ZhangLab 2 2014-04-02 13hr 46min MONOD v3capture gel2.jpg
- Purify PCR product with 0.7 volume of AMPure beads
- Eluted with 60ul H2O
- Quantify concentration in 6% TBE gel (load 2ul of purified libraries)
File:ZhangLab 2 2014-04-02 17hr 21min MONOD V3 Seq PQ 1-8.jpg File:ZhangLab 2 2014-04-02 17hr 21min MONOD V3 Seq PQ 9-13.jpg
Sample ID Original IDs Index Conc. (ng/ul) Total amount in 58ul (ng) Volume for 50ng (ul)
MN_1 BXPC3 Ind_1 1.41 81.93 35.39
MN_2 U87MG Ind_2 1.12 64.93 44.67
MN_3 PANC1 Ind_3 1.67 97.11 29.86
MN_4 BE(2)C Ind_4 3.13 181.61 15.97
MN_5 T98G Ind_5 3.74 216.67 13.38
MN_6 H9 p43 BMP4 Ind_6 2.13 123.51 23.48
MN_7 H1 p47 Control Ind_7 2.96 171.40 16.92
MN_8 H1 p47 BMP-4 Ind_8 2.86 165.82 17.49
MN_9 UCLA-B-F10 Ind_9 3.27 189.49 15.30
MN_10 UCLA-B-G10 Ind_10 2.75 159.39 18.19
MN_11 UCLA-B-H11 Ind_11 0.91 52.65 55.08
MN_13 BXPC3_Stoffel Ind_12 2.68 155.23 18.68
- Pool equal amount of sequencing libraries (50ng each) and performed PAGE size-selection in 2 of 5-well 6% TBE gels at the band ~375bp
File:ZhangLab 2 2014-04-09 11hr 37min MONOD V3 SeqLib PAGESS-1.jpg File:ZhangLab 2 2014-04-09 11hr 37min MONOD V3 SeqLib PAGESS-2.jpg
- Elute with 30ul H2O
- Verify sequencing libraries in 6% TBE gel by loading 2ul

Sequencing ibrary IDs: NP-BSPP-MONOD_V3_Mar30, 2014
Sequencing run: PE run, 150+7+150 bp

- MiSeq run: 140411_MiSeq (PE 76 + 6 + 76 in stead of PE 150+7+150 bp)