Noi/NOTES/2014-8-3: Difference between revisions
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>Noi mNo edit summary |
>Noi mNo edit summary |
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| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 1ng''' | | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 1ng''' | ||
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O''' | | align="center" style="width:80px;background:#f0f0f0;"|'''H2O''' | ||
| align="center" style="width:80px;background:#f0f0f0;"|'''Klenow,exo-: | | align="center" style="width:80px;background:#f0f0f0;"|'''Klenow,exo-:dAdCdG Mix''' | ||
| align="center" style="width:80px;background:#f0f0f0;"|'''Total''' | | align="center" style="width:80px;background:#f0f0f0;"|'''Total''' | ||
| align="center" style="width:60px;background:#f0f0f0;"|'''# in strip''' | | align="center" style="width:60px;background:#f0f0f0;"|'''# in strip''' | ||
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|- | |- | ||
| NTC_2||0.000||0.00||10.00||10.00||20.00||NA||ntc2 | | NTC_2||0.000||0.00||10.00||10.00||20.00||NA||ntc2 | ||
|- | |||
| JURKAT_1||1.000||1||9.00||10.00||20.00||NA||JK_1 | |||
|- | |||
| JURKAT_1||1.000||1.00||9.00||10.00||20.00||NA||JK_2 | |||
|} | |} | ||
==== <u>Klenow exo- and dA:dG:dC reaction mix</u> ==== | ==== <u>Klenow exo- and dA:dG:dC reaction mix</u> ==== | ||
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| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | ||
| align="center" style="width:80px;background:#f0f0f0;"|''' | | align="center" style="width:80px;background:#f0f0f0;"|'''30x rxn''' | ||
|- | |- | ||
| 10X Tango buffer||2.00|| | | 10X Tango buffer||2.00||60.00 | ||
|- | |- | ||
| dA:dC:dG (20:2:2mM)||1.00|| | | dA:dC:dG (20:2:2mM)||1.00||30.00 | ||
|- | |- | ||
| Klenow fragment, exo- (5U/ul)||1.00|| | | Klenow fragment, exo- (5U/ul)||1.00||30.00 | ||
|- | |- | ||
| H2O||6.00|| | | H2O||6.00||180.00 | ||
|- | |- | ||
| Total||10.00|| | | Total||10.00|| | ||
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== 2) Methylated adaptor ligation == | == 2) Methylated adaptor ligation == | ||
* Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 25ul | * Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 25ul | ||
{| {{table}} class=wikitable | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Exp ID''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''TruSeq Index''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Exp ID''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''TruSeq Index''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Exp ID''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''TruSeq Index''' | |||
|- | |||
| wPC-P-2||1||w6-P-1||13||w7-P-1||11 | |||
|- | |||
| wPC-P-3||2||w6-P-2||14||w7-P-2||12 | |||
|- | |||
| wPC-P-4||4||w6-P-3||15||w7-P-3||21 | |||
|- | |||
| wPC-P-5||5||w6-P-4||16||w7-P-5||22 | |||
|- | |||
| wPC-P-6||6||w6-P-5||27||w7-P-6||3 | |||
|- | |||
| wPC-P-7||7||w6-P-6||18||w7-P-7||9 | |||
|- | |||
| wPC-P-8||8||w6-P-8||19||w7-P-8||23 | |||
|- | |||
| wPC-P-9||10||w6-P-9||20||w7-P-10||25 | |||
|} | |||
==== <u>Ligation reaction mix</u> ==== | ==== <u>Ligation reaction mix</u> ==== | ||
{| {{table}} border = 1 | {| {{table}} border = 1 | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | ||
| align="center" style="width:80px;background:#f0f0f0;"|''' | | align="center" style="width:80px;background:#f0f0f0;"|'''30x rxn''' | ||
|- | |- | ||
| dA-tailed DNA||20.00||0.00 | | dA-tailed DNA||20.00||0.00 | ||
|- | |- | ||
| 10X Tango buffer||0.50|| | | 10X Tango buffer||0.50||15.00 | ||
|- | |- | ||
| HC T4 DNA ligase (30units/ul)||1.00|| | | HC T4 DNA ligase (30units/ul)||1.00||30.00 | ||
|- | |- | ||
| 10mM ATP ||1.25|| | | 10mM ATP ||1.25||37.50 | ||
|- | |- | ||
| H2O||1.25|| | | H2O||1.25||37.50 | ||
|- | |- | ||
| Total||24.00|| | | Total||24.00|| |
Revision as of 00:31, 4 August 2014
Preparation of WGBS libraries of small fragment DNA in plasma samples (no DNA fragmentation step)
Select plasma DNA samples for WGBS
Pancreas (UCSD-004)
Diagnosis | Conc. (ng/uL) | Volume (ul) | Yields (ng) | Selected for WGBS | Zhang lab ID |
Adenocarcinoma, moderately to poorly differentiated, c/w pT3N0MX | 0.473 | 30.00 | 14.19 | PC-P-1 | |
Adenocarcinoma, ductal type, moderately to poorly differentiated, pT3N1MX | 0.852 | 30.00 | 25.56 | YES | PC-P-2 |
Poorly differentiated adenocarcinoma, ductal type, pT3N1MX | 1.360 | 30.00 | 40.80 | YES | PC-P-3 |
Adenocarcinoma, ductal type, moderately to poorly differentiated, involving head and body of pancreas and invading into duodenal submucosa and peripancreatic soft tissue, c/w pT3N1MX | 0.340 | 30.00 | 10.20 | YES | PC-P-4 |
Poorly differentiated pancreatic ductal adenocarcinoma, invading into duodenum, consistent with pT3N0MX | 0.813 | 30.00 | 24.39 | YES | PC-P-5 |
Invasive pancreatic duct adenocarcinoma, moderately to poorly differentiated, Stage: ypT3N1MX, Stage IIB | 0.767 | 30.00 | 23.01 | YES | PC-P-6 |
Invasice ductal adenocarcinoma of the pancreas, moderately to poorly differentiated, T3N1MX, stage IIB | 0.644 | 30.00 | 19.32 | YES | PC-P-7 |
Metastatic pancreatic adenocarcinoma | 0.524 | 30.00 | 15.72 | YES | PC-P-8 |
Metastatic adenocarcinoma | 0.238 | 30.00 | 7.14 | YES | PC-P-9 |
Adenocarcinoma, moderately to poorly differentiated, c/w pT3N0MX | 0.370 | 30.00 | 11.10 | PC-P-10 |
Colon (UCSD-006)
Diagnosis | Conc. (ng/uL) | Volume (ul) | Yields (ng) | Selected for WGBS | Zhang lab ID |
Poorly differentiated adenocarcinoma, consistent with recurrent colonic adenocarcinoma | 0.200 | 30.00 | 6.00 | YES | 6-P-1 |
Metastatic malignant hemangiopericytoma/ malignant solitary fibrous tumor | 1.100 | 30.00 | 33.00 | YES | 6-P-2 |
Sigmoid colon, rectum, and anus, resection. Recurrent invasive moderately- to poorly- differentiated | 1.570 | 30.00 | 47.10 | YES | 6-P-3 |
Metastatic adenocarcinoma. | 0.889 | 30.00 | 26.67 | YES | 6-P-4 |
Metastatic mucinous adenocarcinoma. | 0.487 | 30.00 | 14.61 | YES | 6-P-5 |
Metastatic moderately-differentiated adenocarcinoma with mucinous features. | 0.249 | 30.00 | 7.47 | YES | 6-P-6 |
Adenocarcinoma | 0.247 | 30.00 | 7.41 | 6-P-7 | |
Metastatic moderately-differentiated adenocarcinoma with municous features | 0.226 | 30.00 | 6.78 | YES | 6-P-8 |
Adenocarcinoma with municous features, moderately differentiated, pT4aN2b | 0.408 | 30.00 | 12.24 | YES | 6-P-9 |
Mucinous adenocarcinoma, moderately differentiated | 0.333 | 30.00 | 9.99 | 6-P-10 |
Lung (UCSD-007)
Diagnosis | Conc. (ng/uL) | Volume (ul) | Yields (ng) | Selected for WGBS | Zhang lab ID |
Invasive moderately differentiated adenocarcinoma, pT26N0 | 0.692 | 30.00 | 20.76 | YES | 7-P-1 |
Poorly differentiated squamous cell carcinoma. Consistent with pT2N0MX, stage IB | 1.140 | 30.00 | 34.20 | YES | 7-P-2 |
Metastatic melanoma | 0.208 | 30.00 | 6.24 | YES | 7-P-3 |
Squamous cell carcinoma, clear cell variant, moderately differentiated | 0.202 | 30.00 | 6.06 | 7-P-4 | |
Squamous cell carcinoma, pT1bN0 | 1.110 | 30.00 | 33.30 | YES | 7-P-5 |
Adenocarcinoma, mix subtype, including 60% bronchioalveolar carcinoma, mucinous subtype with 40% invasive carcinoma, acinar and papillary subtypes | 0.233 | 30.00 | 6.99 | YES | 7-P-6 |
Invasive squamous carcinoma, moderately differentiated, consistent with pT1N0MX | 0.229 | 30.00 | 6.87 | YES | 7-P-7 |
Invasive adenocarcinoma, poorly differentiated | 0.167 | 30.00 | 5.01 | YES | 7-P-8 |
Non-small-cell carcinoma | 0.231 | 30.00 | 6.93 | 7-P-9 | |
Poorly differentiated carcinoma, possibly adenoSCC | 0.203 | 30.00 | 6.09 | YES | 7-P-10 |
1) End-repair/dA-tailing
- Incubate plasma DNA with 5U Klenow fragment, exo- and dA:dC:dG (final conc. 1:0.1:0.1mM) in total volume 20ul
Pancreas
Zhang lab ID | Conc. (ng/ul) | Volume for 1ng | H2O | Klenow,exo-:dA:dC:dG Mix | Total | # in strip | Exp ID |
PC-P-2 | 0.852 | 1.17 | 8.83 | 10.00 | 20.00 | #4_1 | wPC-P-2 |
PC-P-3 | 1.360 | 0.74 | 9.26 | 10.00 | 20.00 | #4_2 | wPC-P-3 |
PC-P-4 | 0.340 | 2.94 | 7.06 | 10.00 | 20.00 | #4_3 | wPC-P-4 |
PC-P-5 | 0.813 | 1.23 | 8.77 | 10.00 | 20.00 | #4_4 | wPC-P-5 |
PC-P-6 | 0.767 | 1.30 | 8.70 | 10.00 | 20.00 | #4_5 | wPC-P-6 |
PC-P-7 | 0.644 | 1.55 | 8.45 | 10.00 | 20.00 | #4_6 | wPC-P-7 |
PC-P-8 | 0.524 | 1.91 | 8.09 | 10.00 | 20.00 | #4_7 | wPC-P-8 |
PC-P-9 | 0.238 | 4.20 | 5.80 | 10.00 | 20.00 | #4_8 | wPC-P-9 |
Colon
Zhang lab ID | Conc. (ng/ul) | Volume for 1ng | H2O | Klenow,exo-:dA:dC:dG Mix | Total | # in strip | Exp ID |
6-P-1 | 0.200 | 5.00 | 5.00 | 10.00 | 20.00 | #6_1 | w6-P-1 |
6-P-2 | 1.100 | 0.91 | 9.09 | 10.00 | 20.00 | #6_2 | w6-P-2 |
6-P-3 | 1.570 | 0.64 | 9.36 | 10.00 | 20.00 | #6_3 | w6-P-3 |
6-P-4 | 0.889 | 1.12 | 8.88 | 10.00 | 20.00 | #6_4 | w6-P-4 |
6-P-5 | 0.487 | 2.05 | 7.95 | 10.00 | 20.00 | #6_5 | w6-P-5 |
6-P-6 | 0.249 | 4.02 | 5.98 | 10.00 | 20.00 | #6_6 | w6-P-6 |
6-P-8 | 0.226 | 4.42 | 5.58 | 10.00 | 20.00 | #6_7 | w6-P-8 |
6-P-9 | 0.408 | 2.45 | 7.55 | 10.00 | 20.00 | #6_8 | w6-P-9 |
Lung
Zhang lab ID | Conc. (ng/ul) | Volume for 1ng | H2O | Klenow,exo-:dAdCdG Mix | Total | # in strip | Exp ID |
7-P-1 | 0.692 | 1.45 | 8.55 | 10.00 | 20.00 | #7_1 | w7-P-1 |
7-P-2 | 1.140 | 0.88 | 9.12 | 10.00 | 20.00 | #7_2 | w7-P-2 |
7-P-3 | 0.208 | 4.81 | 5.19 | 10.00 | 20.00 | #7_3 | w7-P-3 |
7-P-5 | 1.110 | 0.90 | 9.10 | 10.00 | 20.00 | #7_4 | w7-P-5 |
7-P-6 | 0.233 | 4.29 | 5.71 | 10.00 | 20.00 | #7_5 | w7-P-6 |
7-P-7 | 0.229 | 4.37 | 5.63 | 10.00 | 20.00 | #7_6 | w7-P-7 |
7-P-8 | 0.167 | 5.99 | 4.01 | 10.00 | 20.00 | #7_7 | w7-P-8 |
7-P-10 | 0.203 | 4.93 | 5.07 | 10.00 | 20.00 | #7_8 | w7-P-10 |
NTC_1 | 0.000 | 0.00 | 10.00 | 10.00 | 20.00 | NA | ntc1 |
NTC_2 | 0.000 | 0.00 | 10.00 | 10.00 | 20.00 | NA | ntc2 |
JURKAT_1 | 1.000 | 1 | 9.00 | 10.00 | 20.00 | NA | JK_1 |
JURKAT_1 | 1.000 | 1.00 | 9.00 | 10.00 | 20.00 | NA | JK_2 |
Klenow exo- and dA:dG:dC reaction mix
Components | 1x rxn | 30x rxn |
10X Tango buffer | 2.00 | 60.00 |
dA:dC:dG (20:2:2mM) | 1.00 | 30.00 |
Klenow fragment, exo- (5U/ul) | 1.00 | 30.00 |
H2O | 6.00 | 180.00 |
Total | 10.00 |
-Aliquot 32.50 of Klwnow, exo- and dA:dC:dG reaction mix to 8-tube strip
- - Add H2O to each tube
- - Add 1ng of DNA to each tube
- - Add 10ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of DNA with multichannel pipette. Pipette few times
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- - 75C, 10min --> hold at 4C (heat lid on)
- No AMPure bead purification
2) Methylated adaptor ligation
- Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 25ul
Exp ID | TruSeq Index | Exp ID | TruSeq Index | Exp ID | TruSeq Index |
wPC-P-2 | 1 | w6-P-1 | 13 | w7-P-1 | 11 |
wPC-P-3 | 2 | w6-P-2 | 14 | w7-P-2 | 12 |
wPC-P-4 | 4 | w6-P-3 | 15 | w7-P-3 | 21 |
wPC-P-5 | 5 | w6-P-4 | 16 | w7-P-5 | 22 |
wPC-P-6 | 6 | w6-P-5 | 27 | w7-P-6 | 3 |
wPC-P-7 | 7 | w6-P-6 | 18 | w7-P-7 | 9 |
wPC-P-8 | 8 | w6-P-8 | 19 | w7-P-8 | 23 |
wPC-P-9 | 10 | w6-P-9 | 20 | w7-P-10 | 25 |
Ligation reaction mix
Components | 1x rxn | 30x rxn |
dA-tailed DNA | 20.00 | 0.00 |
10X Tango buffer | 0.50 | 15.00 |
HC T4 DNA ligase (30units/ul) | 1.00 | 30.00 |
10mM ATP | 1.25 | 37.50 |
H2O | 1.25 | 37.50 |
Total | 24.00 |
- - Aliquot 13ul of ligation reaction mix to 8-tube strip
- - Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
- - Add 4ul of ligation reaction mix with multichannel pipette
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 16C, 20h (no heat lid)
- - Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- - Continue to bisulfite conversion