Noi/NOTES/2014-8-3: Difference between revisions

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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-:dA:dC:dG Mix'''
| 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'''
Line 175: Line 175:
|-
|-
| 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;"|'''28x rxn'''
| align="center" style="width:80px;background:#f0f0f0;"|'''30x rxn'''
|-
|-
| 10X Tango buffer||2.00||56.00
| 10X Tango buffer||2.00||60.00
|-
|-
| dA:dC:dG (20:2:2mM)||1.00||28.00
| dA:dC:dG (20:2:2mM)||1.00||30.00
|-
|-
| Klenow fragment, exo- (5U/ul)||1.00||28.00
| Klenow fragment, exo- (5U/ul)||1.00||30.00
|-
|-
| H2O||6.00||168.00
| H2O||6.00||180.00
|-
|-
| Total||10.00||
| Total||10.00||
Line 205: Line 209:
== 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;"|'''28x rxn'''
| 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||14.00
| 10X Tango buffer||0.50||15.00
|-
|-
| HC T4 DNA ligase (30units/ul)||1.00||28.00
| HC T4 DNA ligase (30units/ul)||1.00||30.00
|-
|-
| 10mM ATP ||1.25||35.00
| 10mM ATP ||1.25||37.50
|-
|-
| H2O||1.25||35.00
| 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)

Calendar

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