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 40ulHemo 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