Noi/NOTES/2011-9-16

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BSPP DMR330k capture of samples from Sergio, two largest pedigrees from HAPMAPPT01, nine replicates, HFF somatic lines, repeat UPenn samples, and comparing DNA methylation kit from Zymoresearch and Sigma[edit]

  • Total number of reactions = 39 reactions
    • 9 samples from Sergio
      • 1. H9 P43 mTeSR
      • 2. H9 p43+Activin 100ng/mL 5days
      • 3. H9 p43+BMP4 50ng/mL 5days
      • 4. HUViPS4F1 p19
      • 5. HUViPS4F1 p19+Activin 100ng/mL 5days
      • 6. HUViPS4F1 p19+BMP4 50ng/mL 5days
      • 7. HUViPS4F3
      • 8. HUViPS4F3+Activin 100ng/mL 5days
      • 9. HUViPS4F3+BMP4 50ng/mL 5days
    • 12 samples from 2 largest pedigrees of HAPMAPPT01
      • Note:
        • wellID = wellID from original HAPMAPPT01 plate
        • All GM IDs (cell products) should be changed to NA (DNA products)
      • Family 1362
        • GM11995 (wellID G7)
        • GM11993 (wellID F2)
        • GM10861 (wellID A9)
        • GM11992 (wellID C2 )
        • GM11994 (wellID G9 )
        • GM10860 (wellID F10)
      • Family 1454
        • GM12813 (wellID E8)
        • GM12802 (wellID E7)
        • GM12815 (wellID G12)
        • GM12812 (wellID F11)
        • GM12814 (wellID A7)
        • GM12801 (wellID D9)
    • 10 replicates
      • NA12156 (HAPMAPPT01, wellID B3)
    • 2 somatic lines
      • 2HFF P6
      • 218 HFF P7
    • 4 samples from Penn (repeat the low QC value samples from the first batch)
    • 2 reactions to compare bis-cvt gDNA quality processed by two different DNA methylation kits, Zymoresearch (DNA methylation gold kit) and Sigma (Imprint DNA modification kit)
      • PGP1iPS
  • Note: For the gDNA of HAPMAPPT01, aliquot 10 ug to each of two working plates and save the original plate

Bisulfite Conversion[edit]

  • Used EZ-96 DNA Methylation-Gold™ Kit (deep well format) Cat#D5008

2011_09_17

  • Note:
    • I just realized that once I got the the first batch Penn samples, I aliquoted DNA sample to 20ul (plate1) and 30 ul (plate2). I still have the samples I understand that I used up.
    • I will used "plate" for the Penn samples stored in the lab and "tube" for the samples I just got from Tishkoff's lab
    • I also got three additional samples from Alice (from Yang Xu's lab) including, Hues1, Hues3 and Hues8 in cell pellets. After extracted gDNA and eluted 150ul each for elute1 and elute2, the concentration was measure by Nanodrop.

Plate layout of bisulfite conversion

Plate layout 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
A Sergio1 Sergio9 GM12802 (wellID E7) NA12156 (wellID B3)-4 218 HFF P7 PGP1iPS
B Sergio2 GM11995 (wellID G7) GM12815 (wellID G12) NA12156 (wellID B3)-5 KEBR028A (B4-plate2)-1 KEBR028A (B4-plate2)-2
C Sergio3 GM11993 (wellID F2) GM12812 (wellID F11) NA12156 (wellID B3)-6 KEBR028A (tube)-1 KEBR028A (tube)-2 Hues1 Hues1
D Sergio4 GM10861 (wellID A9) GM12814 (wellID A7) NA12156 (wellID B3)-7 KEBR040 (D4-plate1) KEBR040 (D4-plate2) Hues3 Hues3
E Sergio5 GM11992 (wellID C2 ) GM12801 (wellID D9) NA12156 (wellID B3)-8 TZHZ018 (F1-plate1) TZHZ018 (F1-plate2) Hues8 Hues8
F Sergio6 GM11994 (wellID G9 ) NA12156 (wellID B3)-1 NA12156 (wellID B3)-9 TZHZ031A (tube)-1 TZHZ031A (tube)-2
G Sergio7 GM10860 (wellID F10) NA12156 (wellID B3)-2 NA12156 (wellID B3)-10 TZHZ041A (tube)-1 TZHZ041A (tube)-2
H Sergio8 GM12813 (wellID E8) NA12156 (wellID B3)-3 2HFF P6 TZHZ075 (H01-plate2)-1 TZHZ075 (H01-plate2)-1


Order Sample ID Family ng/ul Volume for 1000ng Well ID Sample ID H2O Total amount (ng) CT conversion reagent (ul)
1 1. H9 62.80 15.92 A1 1. H9 4.08 1000.00 130.00
2 2. H9+Activin 100ng/mL 5days 90.80 11.01 B1 2. H9+Activin 100ng/mL 5days 8.99 1000.00 130.00
3 3. H9+BMP4 50ng/mL 5days 93.70 10.67 C1 3. H9+BMP4 50ng/mL 5days 9.33 1000.00 130.00
4 4. HUViPS4F1 98.00 10.20 D1 4. HUViPS4F1 9.80 1000.00 130.00
5 5. HUViPS4F1+Activin 100ng/mL 5days 84.80 11.79 E1 5. HUViPS4F1+Activin 100ng/mL 5days 8.21 1000.00 130.00
6 6. HUViPS4F1+BMP4 50ng/mL 5days 78.60 12.72 F1 6. HUViPS4F1+BMP4 50ng/mL 5days 7.28 1000.00 130.00
7 7. HUViPS4F3 96.90 10.32 G1 7. HUViPS4F3 9.68 1000.00 130.00
8 8. HUViPS4F3+Activin 100ng/mL 5days 91.90 10.88 H1 8. HUViPS4F3+Activin 100ng/mL 5days 9.12 1000.00 130.00
9 9. HUViPS4F3+BMP4 50ng/mL 5days 80.60 12.41 A3 9. HUViPS4F3+BMP4 50ng/mL 5days 7.59 1000.00 130.00
10 GM11995 (wellID G7) 1362 200.00 5.00 B3 GM11995 (wellID G7) 15.00 1000.00 130.00
11 GM11993 (wellID F2) 1362 200.00 5.00 C3 GM11993 (wellID F2) 15.00 1000.00 130.00
12 GM10861 (wellID A9) 1362 200.00 5.00 D3 GM10861 (wellID A9) 15.00 1000.00 130.00
13 GM11992 (wellID C2 ) 1362 200.00 5.00 E3 GM11992 (wellID C2 ) 15.00 1000.00 130.00
14 GM11994 (wellID G9 ) 1362 200.00 5.00 F3 GM11994 (wellID G9 ) 15.00 1000.00 130.00
15 GM10860 (wellID F10) 1362 200.00 5.00 G3 GM10860 (wellID F10) 15.00 1000.00 130.00
16 GM12813 (wellID E8) 1454 200.00 5.00 H3 GM12813 (wellID E8) 15.00 1000.00 130.00
17 GM12802 (wellID E7) 1454 200.00 5.00 A5 GM12802 (wellID E7) 15.00 1000.00 130.00
18 GM12815 (wellID G12) 1454 200.00 5.00 B5 GM12815 (wellID G12) 15.00 1000.00 130.00
19 GM12812 (wellID F11) 1454 200.00 5.00 C5 GM12812 (wellID F11) 15.00 1000.00 130.00
20 GM12814 (wellID A7) 1454 200.00 5.00 D5 GM12814 (wellID A7) 15.00 1000.00 130.00
21 GM12801 (wellID D9) 1454 200.00 5.00 E5 GM12801 (wellID D9) 15.00 1000.00 130.00
22 NA12156 (wellID B3)-1 1408 200.00 5.00 F5 NA12156 (wellID B3)-1 15.00 1000.00 130.00
23 NA12156 (wellID B3)-2 1408 200.00 5.00 G5 NA12156 (wellID B3)-2 15.00 1000.00 130.00
24 NA12156 (wellID B3)-3 1408 200.00 5.00 H5 NA12156 (wellID B3)-3 15.00 1000.00 130.00
25 NA12156 (wellID B3)-4 1408 200.00 5.00 A7 NA12156 (wellID B3)-4 15.00 1000.00 130.00
26 NA12156 (wellID B3)-5 1408 200.00 5.00 B7 NA12156 (wellID B3)-5 15.00 1000.00 130.00
27 NA12156 (wellID B3)-6 1408 200.00 5.00 C7 NA12156 (wellID B3)-6 15.00 1000.00 130.00
28 NA12156 (wellID B3)-7 1408 200.00 5.00 D7 NA12156 (wellID B3)-7 15.00 1000.00 130.00
29 NA12156 (wellID B3)-8 1408 200.00 5.00 E7 NA12156 (wellID B3)-8 15.00 1000.00 130.00
30 NA12156 (wellID B3)-9 1408 200.00 5.00 F7 NA12156 (wellID B3)-9 15.00 1000.00 130.00
31 NA12156 (wellID B3)-10 1408 200.00 5.00 G7 NA12156 (wellID B3)-10 15.00 1000.00 130.00
32 2HFF P6 280.10 3.57 H7 2HFF P6 16.43 1000.00 130.00
33 218 HFF P7 446.10 2.24 A9 218 HFF P7 17.76 1000.00 130.00
34 KEBR028A (B4-plate2)-1,2 170.70 5.86 B9,B10 KEBR028A (B4-plate2)-1 14.14 1000.00 130.00
35 KEBR028A (tube)-1,2 23.80 20.00 C9,C10 KEBR028A (tube)-1 0.00 476.00 130.00
36 KEBR040 (D4-plate1) 173.50 5.76 D9 KEBR040 (D4-plate1) 14.24 1000.00 130.00
KEBR040 (D4-plate2) 111.50 8.97 D10 KEBR040 (D4-plate2) 11.03 1000.00 130.00
37 TZHZ018 (F1-plate1) 185.20 5.40 E9 TZHZ018 (F1-plate1) 14.60 1000.00 130.00
TZHZ018 (F1-plate2) 196.10 5.10 E10 TZHZ018 (F1-plate2) 14.90 1000.00 130.00
38 TZHZ031A (tube)-1,2 55.10 18.15 F9,F10 TZHZ031A (tube)-1 1.85 1000.00 130.00
39 TZHZ041A (tube)-1,2 54.60 18.32 G9,G10 TZHZ041A (tube)-1 1.68 1000.00 130.00
40 TZHZ075 (H01-plate2)-1,2 58.10 17.21 H9,H10 TZHZ075 (H01-plate2)-1 2.79 1000.00 130.00
41 PGP1iPS 129.00 7.75 A11 PGP1iPS 12.25 1000.00 130.00
42 Hues1 341.90 2.92 C11,C12 Hues1 17.08 1000.00 130.00
43 Hues3 696.90 1.43 D11,D12 Hues3 18.57 1000.00 130.00
44 Hues8 410.60 2.44 E11,E12 Hues8 17.56 1000.00 130.00
  • Eluted volume 20ul with Elution buffer. After Qubit ss-DNA assay the total yields will be calculated based on ~17ul left in the well (or a little bit lower).

Qubit ssDNA assay[edit]

  • Follow protocol provided with the kit
  • Took 1ul of bis-cvt DNA into 9ul of H2O, then added the diluted DNA to 190ul ssDNA Qubit reagent (dilution factor = 200)
Sample ID Well ID Concentration in the Qubit (ng/mL) Volume (ul) Final conc. (ng/ul) Yields (ng)
1. H9 A1 76.40 17.00 15.28 252.12
**2. H9+Activin 100ng/mL 5days B1 62.70 17.00 12.54 206.91
3. H9+BMP4 50ng/mL 5days C1 110.00 17.00 22.00 363.00
4. HUViPS4F1 D1 117.00 17.00 23.40 386.10
5. HUViPS4F1+Activin 100ng/mL 5days E1 76.90 17.00 15.38 253.77
6. HUViPS4F1+BMP4 50ng/mL 5days F1 114.00 17.00 22.80 376.20
7. HUViPS4F3 G1 118.00 17.00 23.60 389.40
8. HUViPS4F3+Activin 100ng/mL 5days H1 79.30 17.00 15.86 261.69
9. HUViPS4F3+BMP4 50ng/mL 5days A3 98.90 17.00 19.78 326.37
GM11995 (wellID G7) B3 205.00 17.00 41.00 676.50
GM11993 (wellID F2) C3 80.80 17.00 16.16 266.64
**GM10861 (wellID A9) D3 51.80 17.00 10.36 170.94
GM11992 (wellID C2 ) E3 112.00 17.00 22.40 369.60
GM11994 (wellID G9 ) F3 138.00 17.00 27.60 455.40
GM10860 (wellID F10) G3 116.00 17.00 23.20 382.80
GM12813 (wellID E8) H3 176.00 17.00 35.20 580.80
GM12802 (wellID E7) A5 133.00 17.00 26.60 438.90
GM12815 (wellID G12) B5 140.00 17.00 28.00 462.00
GM12812 (wellID F11) C5 112.00 17.00 22.40 369.60
GM12814 (wellID A7) D5 97.10 17.00 19.42 320.43
GM12801 (wellID D9) E5 122.00 17.00 24.40 402.60
NA12156 (wellID B3)-1 F5 72.20 17.00 14.44 238.26
NA12156 (wellID B3)-2 G5 68.40 17.00 13.68 225.72
NA12156 (wellID B3)-3 H5 76.30 17.00 15.26 251.79
NA12156 (wellID B3)-4 A7 85.30 17.00 17.06 281.49
NA12156 (wellID B3)-5 B7 81.00 17.00 16.20 267.30
NA12156 (wellID B3)-6 C7 85.20 17.00 17.04 281.16
NA12156 (wellID B3)-7 D7 65.70 17.00 13.14 216.81
NA12156 (wellID B3)-8 E7 80.10 17.00 16.02 264.33
NA12156 (wellID B3)-9 F7 69.30 17.00 13.86 228.69
NA12156 (wellID B3)-10 G7 68.90 17.00 13.78 227.37
**2HFF P6 H7 44.00 17.00 8.80 145.20
**218 HFF P7 A9 41.20 17.00 8.24 135.96
KEBR028A (B4-plate2)-1,2 B9 38.50 17.00 7.70 127.05
****KEBR028A (tube)-1,2 C9 Out Of Range 17.00 #VALUE! #VALUE!
KEBR040 (D4-plate1) D9 157.00 17.00 31.40 518.10
TZHZ018 (F1-plate1) E9 137.00 17.00 27.40 452.10
TZHZ031A (tube)-1,2 F9 274.00 17.00 54.80 904.20
TZHZ041A (tube)-1,2 G9 215.00 17.00 43.00 709.50
TZHZ075 (H01-plate2)-1,2 H9 164.00 17.00 32.80 541.20
**PGP1iPS A11 30.00 17.00 6.00 99.00
PGP1iPS-IMPRINT B11 42.90 17.00 8.58 141.57
**Hues1 C11 35.80 17.00 7.16 118.14
**Hues3 D11 34.60 17.00 6.92 114.18
**Hues8 E11 43.40 17.00 8.68 143.22
  • Continued on 2011_09_18: [[1]]