Noi/NOTES/2012-5-20

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DMR 330k BSPP capture of samples from Sergio[edit]

Sample list[edit]

New gDNA extracted from the cell pellet[edit]

on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-17
No. Sample ID Lab ID Conc. (ng/ul) Volume (ul) Yields (ug)
1 HUES2 p41 Ap15-1 110.0 250 27.5
2 ASThiPS4F1 p12 Ap15-2 139.1 250 34.8
3 ASThiPS4F2 p12 Ap15-3 145.1 250 36.3
4 HUES6 p39 Control TeSR Ap15-4 295.5 250 73.9
5 HUES6 p39 ACTIVIN Ap15-5 128.1 250 32.0
6 HUES6 p39 BMP-4 Ap15-6 153.9 250 38.5
7 HUES9 p39 Control TeSR Ap15-7 183.5 250 45.9
8 HUES9 p39 ACTIVIN Ap15-8 149.3 250 37.3
9 HUES9 p39 BMP-4 Ap15-9 111.2 250 27.8
10 HUES3 p36 Control TeSR Ap15-10 153.5 250 38.4
11 HUES3 p36 ACTIVIN Ap15-11 87.5 250 21.9
12 HUES3 p36 BMP-4 Ap15-12 109.7 250 27.4
13 H1 p54 Control TeSR Ap15-13 191.3 250 47.8
14 H1 p54 ACTIVIN Ap15-14 108.7 250 27.2
15 ASThiPS4F5 p16 Control TeSR Ap15-15 134.2 250 33.6
*16 ASThiPS4F5 p16 ACTIVIN Ap15-16 50.2 500 25.1
17 ASThiPS4F4 p36 Control TeSR Ap15-17 183.0 250 45.8
18 ASThiPS4F4 p36 ACTIVIN Ap15-18 82.6 250 20.7
19 ASThiPS4F4 p36 BMP-4 Ap15-19 263.4 250 65.9
20 H1 p48 Ma15-2 215.2 200 43.0
21 H9 p47 Ma15-3 252.0 200 50.4
22 HUES3 p33 Ma15-4 226.1 200 45.2
23 HUES6 p26 Ma15-5 238.7 200 47.7
24 HUES8 p35 Ma15-6 83.4 200 16.7
25 HUES9 p36 Ma15-7 236.8 200 47.4
26 ASThiPS4F3 p24 (derived from Astrocytes) Ma15-8 175.9 200 35.2
27 ASThiPS4F4 p16 (derived from Astrocytes) Ma15-9 164.3 200 32.9
28 FiPS4F2 p32 (derived from IMR90 fibroblasts) Ma15-10 205.9 200 41.2
29 FiPS4F5 p29 (derived from IMR90 fibroblasts) Ma15-11 97.6 200 19.5
30 KiPS4FA p54 (derived from keratinocytes) Ma15-12 129.5 200 25.9
31 Huv-iPS4F6 p23 (derived from HUVECs) Ma15-13 263.1 200 52.6
32 IMR90 p6 Ma15-14 76.1 200 15.2
33 Keratinocytes p3 Ma15-15 138.7 200 27.7
34 H1 p47 Control Ma15-16 216.4 200 43.3
35 H1 p47 BMP-4 Ma15-17 137.6 200 27.5
36 ASThiPS4F5 p12 Control Ma15-18 224.6 200 44.9
37 ASThiPS4F5 p12 BMP-4 Ma15-19 111.7 200 22.3
38 Huv-iPS4F6 p23 Control Ma15-20 289.9 200 58.0
39 Huv-iPS4F6 p23 BMP-4 Ma15-21 172.9 200 34.6
40 Huv-iPS4F6 p23 ACTIVIN Ma15-1 86.7 250 21.7

Old gDNA of samples have been captured in the previous experiments[edit]

  • Note:
    • Some of gDNA concentrations were re-measured and note that some of them have conc. lower than 50ng/ul, and some of them don't have enough amount for bisulfite conversion
    • Samples KERAT MMTA and KiPS4F #8 have high conc., but very little volume. I added ~17ul H2O then re-measured conc. again
    • ***I may not do bis-cvt on the two samples, SR-21 and SR22 since the amount of gDNA was too little
No Sample IDs Lab IDs Conc. (ng/ul)
1 H9 p43 mTeSR SR-1 62.8
2 H9 p43 ACTIVIN SR-2 90.8
3 H9 p43 BMP4 SR-3 93.7
4 HUV0iPS4F1 p19 SR-4 98.0
5 HUV-hiPS4F1 p20 ACTIVIN SR-5 84.8
6 HUV-hiPS4F1 p20 BMP4 SR-6 78.6
7 HUV-iPS4F3 F19 SR-7 96.9
8 HUV-hiPS4F3 p20 ACTIVIN SR-8 91.9
9 HUV-hiPS4F3 p20 BMP4 SR-9 80.6
10 aMSCs p7 SR-10 184.8
11 MSCiPS #8 p14 (~4 ul) SR-11 105.4
12 MSCiPS #4 p14 SR-12 181.4
13 hNiPS2F p23 SR-13 270.0
14 hKiPS3F-7 p24 (~15ul) SR-14 33.0
15 hNSC SR-15 67.0
16 hFiPS4F-7 p9 SR-16 145.6
17 HUVEC p2 SR-17 245.3
18 hiPS AST4F-4 p9 SR-18 89.9
19 KERAT MMTA SR-19 159.5
20 KiPS4F #8 SR-20 195.7
21 ***olf MSC 19.1ng/ul volume only 3-5ul SR-21 19.1
22 ***KiPS 14ng/ul volume only 3-5ul SR-22 14.0

8 samples of bis-cvt gDNA from Dinh[edit]

  • The list will be filled later and need to re-measure of the conc. with ssDNA Qubit assay

Bisulfite conversion[edit]

Reagent prep
Add the following reagents to CT Conversion Reagent
  -  H2O                   9.0mL
  - M-Dissolving Buffer    500ul
  - M-Dilution Buffer      3.0mL
Mix at room temperature with frequent vortexing or shaking for 15 minutes
Add 144 ml of 100% ethanol to the 36 ml M-Wash Buffer concentrate before use
Lab IDs Conc. (ng/ul) Volume for 1200ng LabID ID Well IDs H2O Total amount (ng) CT conversion reagent (ul) Total volume (ul)
Ap15-1 110.0 10.91 Ap15-1 A1 9.09 1200.00 130.00 150.00
Ap15-2 139.1 8.63 Ap15-2 B1 11.37 1200.00 130.00 150.00
Ap15-3 145.1 8.27 Ap15-3 C1 11.73 1200.00 130.00 150.00
Ap15-4 295.5 4.06 Ap15-4 D1 15.94 1200.00 130.00 150.00
Ap15-5 128.1 9.37 Ap15-5 E1 10.63 1200.00 130.00 150.00
Ap15-6 153.9 7.80 Ap15-6 F1 12.20 1200.00 130.00 150.00
Ap15-7 183.5 6.54 Ap15-7 G1 13.46 1200.00 130.00 150.00
Ap15-8 149.3 8.04 Ap15-8 H1 11.96 1200.00 130.00 150.00
Ap15-9 111.2 10.79 Ap15-9 A2 9.21 1200.00 130.00 150.00
Ap15-10 153.5 7.82 Ap15-10 B2 12.18 1200.00 130.00 150.00
Ap15-11 87.5 13.71 Ap15-11 C2 6.29 1200.00 130.00 150.00
Ap15-12 109.7 10.94 Ap15-12 D2 9.06 1200.00 130.00 150.00
Ap15-13 191.3 6.27 Ap15-13 E2 13.73 1200.00 130.00 150.00
Ap15-14 108.7 11.04 Ap15-14 F2 8.96 1200.00 130.00 150.00
Ap15-15 134.2 8.94 Ap15-15 G2 11.06 1200.00 130.00 150.00
Ap15-16 50.2 20.00 Ap15-16 H2 0.00 1200.00 130.00 150.00
Ap15-17 183.0 6.56 Ap15-17 A3 13.44 1200.00 130.00 150.00
Ap15-18 82.6 14.53 Ap15-18 B3 5.47 1200.00 130.00 150.00
Ap15-19 263.4 4.56 Ap15-19 C3 15.44 1200.00 130.00 150.00
Ma15-1 86.7 13.84 Ma15-1 D3 6.16 1200.00 130.00 150.00
Ma15-2 215.2 5.58 Ma15-2 E3 14.42 1200.00 130.00 150.00
Ma15-3 252.0 4.76 Ma15-3 F3 15.24 1200.00 130.00 150.00
Ma15-4 226.1 5.31 Ma15-4 G3 14.69 1200.00 130.00 150.00
Ma15-5 238.7 5.03 Ma15-5 H3 14.97 1200.00 130.00 150.00
Ma15-6 83.4 14.39 Ma15-6 A4 5.61 1200.00 130.00 150.00
Ma15-7 236.8 5.07 Ma15-7 B4 14.93 1200.00 130.00 150.00
Ma15-8 175.9 6.82 Ma15-8 C4 13.18 1200.00 130.00 150.00
Ma15-9 164.3 7.30 Ma15-9 D4 12.70 1200.00 130.00 150.00
Ma15-10 205.9 5.83 Ma15-10 E4 14.17 1200.00 130.00 150.00
Ma15-11 97.6 12.30 Ma15-11 F4 7.70 1200.00 130.00 150.00
Ma15-12 129.5 9.27 Ma15-12 G4 10.73 1200.00 130.00 150.00
Ma15-13 263.1 4.56 Ma15-13 H4 15.44 1200.00 130.00 150.00
Ma15-14 76.1 15.77 Ma15-14 A5 4.23 1200.00 130.00 150.00
Ma15-15 138.7 8.65 Ma15-15 B5 11.35 1200.00 130.00 150.00
Ma15-16 216.4 5.55 Ma15-16 C5 14.45 1200.00 130.00 150.00
Ma15-17 137.6 8.72 Ma15-17 D5 11.28 1200.00 130.00 150.00
Ma15-18 224.6 5.34 Ma15-18 E5 14.66 1200.00 130.00 150.00
Ma15-19 111.7 10.74 Ma15-19 F5 9.26 1200.00 130.00 150.00
Ma15-20 289.9 4.14 Ma15-20 G5 15.86 1200.00 130.00 150.00
Ma15-21 172.9 6.94 Ma15-21 H5 13.06 1200.00 130.00 150.00
Lab IDs Conc. (ng/ul) Volume for 1200ng LabID ID Well IDs H2O Total amount (ng) CT conversion reagent (ul) Total volume (ul)
SR-1 62.8 19.11 SR-1 A6 0.89 1200.00 130.00 150.00
SR-2 90.8 13.22 SR-2 B6 6.78 1200.00 130.00 150.00
SR-3 93.7 12.81 SR-3 C6 7.19 1200.00 130.00 150.00
SR-4 98.0 12.24 SR-4 D6 7.76 1200.00 130.00 150.00
SR-5 84.8 14.15 SR-5 E6 5.85 1200.00 130.00 150.00
SR-6 78.6 15.27 SR-6 F6 4.73 1200.00 130.00 150.00
SR-7 96.9 12.38 SR-7 G6 7.62 1200.00 130.00 150.00
SR-8 91.9 13.06 SR-8 H6 6.94 1200.00 130.00 150.00
SR-9 80.6 14.89 SR-9 A7 5.11 1200.00 130.00 150.00
SR-10 184.8 6.49 SR-10 B7 13.51 1200.00 130.00 150.00
SR-11 105.4 5.00 SR-11 C7 15.00 527.00 130.00 150.00
SR-12 181.4 6.62 SR-12 D7 13.38 1200.00 130.00 150.00
SR-13 270.0 4.44 SR-13 E7 15.56 1200.00 130.00 150.00
SR-14 33.0 20.00 SR-14 F7 0.00 495.00 130.00 150.00
SR-15 67.0 17.91 SR-15 G7 2.09 1200.00 130.00 150.00
SR-16 145.6 8.24 SR-16 H7 11.76 1200.00 130.00 150.00
SR-17 245.3 4.89 SR-17 A8 15.11 1200.00 130.00 150.00
SR-18 89.9 13.35 SR-18 B8 6.65 1200.00 130.00 150.00
SR-19 159.5 7.52 SR-19 C8 12.48 1200.00 130.00 150.00
SR-20 195.7 6.13 SR-20 D8 13.87 1200.00 130.00 150.00
SR-21 19.1 5.00 SR-21 E8 15.00 95.50 130.00 150.00
SR-22 14.0 5.00 SR-22 F8 15.00 70.00 130.00 150.00
Program
   98°C for 10 minutes
   64°C for 2.5 hours
   4°C storage up to 20 hours
Measure conc. with ssDNA Qubit assay (used 1ul of bis-cvt gDNA)

Qubit ssDNA assay[edit]

  • Sample in assay 1ul, dilution 200x
Sample LabID ID Well IDs Concentration in the Qubit ' Final conc. (ng/ul) Yields in 16ul (ng) LabID ID Well IDs Repeated samples
1.00 Ap15-1 A1 57.00 ng/mL 11.40 182.40 Ap15-1 A1 X
2.00 Ap15-2 B1 62.20 ng/mL 12.44 199.04 Ap15-2 B1 X
3.00 Ap15-3 C1 81.70 ng/mL 16.34 261.44 Ap15-3 C1 X
4.00 Ap15-4 D1 75.40 ng/mL 15.08 241.28 Ap15-4 D1 X
5.00 Ap15-5 E1 59.60 ng/mL 11.92 190.72 Ap15-5 E1 X
6.00 Ap15-6 F1 74.50 ng/mL 14.90 238.40 Ap15-6 F1 X
7.00 Ap15-7 G1 76.40 ng/mL 15.28 244.48 Ap15-7 G1 X
8.00 Ap15-8 H1 76.40 ng/mL 15.28 244.48 Ap15-8 H1 X
9.00 Ap15-9 A2 76.10 ng/mL 15.22 243.52 Ap15-9 A2 X
10.00 Ap15-10 B2 60.10 ng/mL 12.02 192.32 Ap15-10 B2 X
11.00 Ap15-11 C2 56.40 ng/mL 11.28 180.48 Ap15-11 C2 X
12.00 Ap15-12 D2 59.00 ng/mL 11.80 188.80 Ap15-12 D2 X
13.00 Ap15-13 E2 72.50 ng/mL 14.50 232.00 Ap15-13 E2 X
14.00 Ap15-14 F2 74.30 ng/mL 14.86 237.76 Ap15-14 F2 X
15.00 Ap15-15 G2 58.90 ng/mL 11.78 188.48 Ap15-15 G2 X
16.00 Ap15-16 H2 45.10 ng/mL 9.02 144.32 Ap15-16 H2 X
17.00 Ap15-17 A3 76.40 ng/mL 15.28 244.48 Ap15-17 A3 X
18.00 Ap15-18 B3 54.90 ng/mL 10.98 175.68 Ap15-18 B3 X
19.00 Ap15-19 C3 78.90 ng/mL 15.78 252.48 Ap15-19 C3 X
20.00 Ma15-1 D3 44.10 ng/mL 8.82 141.12 Ma15-1 D3 X
21.00 Ma15-2 E3 49.80 ng/mL 9.96 159.36 Ma15-2 E3 X
22.00 Ma15-3 F3 51.60 ng/mL 10.32 165.12 Ma15-3 F3 X
23.00 Ma15-4 G3 58.20 ng/mL 11.64 186.24 Ma15-4 G3 X
24.00 Ma15-5 H3 60.10 ng/mL 12.02 192.32 Ma15-5 H3 X
25.00 Ma15-6 A4 40.60 ng/mL 8.12 129.92 Ma15-6 A4 X
26.00 Ma15-7 B4 59.90 ng/mL 11.98 191.68 Ma15-7 B4 X
27.00 Ma15-8 C4 54.50 ng/mL 10.90 174.40 Ma15-8 C4 X
28.00 Ma15-9 D4 43.60 ng/mL 8.72 139.52 Ma15-9 D4 X
29.00 Ma15-10 E4 73.10 ng/mL 14.62 233.92 Ma15-10 E4 X
30.00 Ma15-11 F4 49.70 ng/mL 9.94 159.04 Ma15-11 F4 X
31.00 Ma15-12 G4 36.90 ng/mL 7.38 118.08 Ma15-12 G4 X
32.00 Ma15-13 H4 60.00 ng/mL 12.00 192.00 Ma15-13 H4 X
33.00 Ma15-14 A5 28.00 ng/mL 5.60 89.60 Ma15-14 A5 X
34.00 Ma15-15 B5 27.70 ng/mL 5.54 88.64 Ma15-15 B5 X
35.00 Ma15-16 C5 53.50 ng/mL 10.70 171.20 Ma15-16 C5 X
36.00 Ma15-17 D5 51.10 ng/mL 10.22 163.52 Ma15-17 D5 X
37.00 Ma15-18 E5 53.60 ng/mL 10.72 171.52 Ma15-18 E5 X
38.00 Ma15-19 F5 51.40 ng/mL 10.28 164.48 Ma15-19 F5 X
39.00 Ma15-20 G5 48.90 ng/mL 9.78 156.48 Ma15-20 G5 X
40.00 Ma15-21 H5 55.40 ng/mL 11.08 177.28 Ma15-21 H5 X
1.00 H9 p43 mTeSR SR-1 108.00 ng/mL 21.60 345.60 SR-1 A6 -
2.00 H9 p43 ACTIVIN SR-2 64.90 ng/mL 12.98 207.68 SR-2 B6 X
3.00 H9 p43 BMP4 SR-3 133.00 ng/mL 26.60 425.60 SR-3 C6 -
4.00 HUV0iPS4F1 p19 SR-4 140.00 ng/mL 28.00 448.00 SR-4 D6 -
5.00 HUV-hiPS4F1 p20 ACTIVIN SR-5 95.40 ng/mL 19.08 305.28 SR-5 E6 -
6.00 HUV-hiPS4F1 p20 BMP4 SR-6 135.00 ng/mL 27.00 432.00 SR-6 F6 -
7.00 HUV-iPS4F3 F19 SR-7 134.00 ng/mL 26.80 428.80 SR-7 G6 -
8.00 HUV-hiPS4F3 p20 ACTIVIN SR-8 96.10 ng/mL 19.22 307.52 SR-8 H6 -
9.00 HUV-hiPS4F3 p20 BMP4 SR-9 120.00 ng/mL 24.00 384.00 SR-9 A7 -
10.00 aMSCs p7 SR-10 53.90 ng/mL 10.78 172.48 SR-10 B7 X
11.00 MSCiPS #8 p14 (~4 ul) SR-11 71.60 ng/mL 14.32 229.12 SR-11 C7 Can\'t repeat
12.00 MSCiPS #4 p14 SR-12 94.60 ng/mL 18.92 302.72 SR-12 D7 -
13.00 hNiPS2F p23 SR-13 29.60 ng/mL 5.92 94.72 SR-13 E7 X
14.00 hKiPS3F-7 p24 (~15ul) SR-14 7.13 ng/mL 1.43 22.82 SR-14 F7 X
15.00 hNSC SR-15 70.90 ng/mL 14.18 226.88 SR-15 G7 X
16.00 hFiPS4F-7 p9 SR-16 23.10 ng/mL 4.62 73.92 SR-16 H7 X
17.00 HUVEC p2 SR-17 50.80 ng/mL 10.16 162.56 SR-17 A8 X
18.00 hiPS AST4F-4 p9 SR-18 131.00 ng/mL 26.20 419.20 SR-18 B8
19.00 KERAT MMTA SR-19 19.80 ng/mL 3.96 63.36 SR-19 C8 X
20.00 KiPS4F #8 SR-20 19.00 ng/mL 3.80 60.80 SR-20 D8 X
21.00 olf MSC 19.1ng/ul volume only 3-5ul SR-21 4.60 ng/mL 0.92 14.72 SR-21 E8 can\'t repeat
22.00 KiPS 14ng/ul volume only 3-5ul SR-22 7.42 ng/mL 1.48 23.74 SR-22 F8 can\'t repeat
  • From the yields of bis-cvt gDNA, many samples have the yields lower than 300ng or 200ng which is the amount the acceptable for capture with BSPP. I checked the yields of the old samples I have processed before (SR-1 - SR-9) to see if this is the technical error of this batch. I got the total yields of SR-1 -15 pretty much the same as I got before. This suggested that there are not the batch effect, but may be the quality of gDNA. I need to repeat bis-cvt again to get enough yield for capture. However, there are some sample that has no more gDNA or has very low concentration, and I don't expected to get higher yield from those samples.