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)