Capturing Bisulfite Converted Jurkat gDNA w GP1 V6 probe set[edit]
- probes prep notes LabNotes/Probe/2014-1-7
[[1]]
Capturing Jurkat gDNA w ppMALT1 ordered form IDT[edit]
Bisulfite Conversion of Jurkat gDNA[edit]
- Bisulfite conversion with zymo methylation gold
- Convert 500ng of Jurkat gDNA (2 rxn, 100ng total)
- Add 130ul of lighting conversion reagent to 5ul of jurkat gDNA and 15ul of ddH2O
- Mix and centrifuge
1. 98°C for 8 minutes
2. 54°C for 60 minutes
3. 4°C hold
- Add 600ul M-binding buffer to column
- load the sample into column, mix by inverting column and centrifuge at 14000rpm for 30sec
- discard flow through
- add 100ul m-wash buffer to the column and centrifuge at 14000rpm for 30sec
- add 200ul L-Desulphonation buffer to the column and sit at RT for 20 minutes and centrifuge at 14000rpm for 30sec
- add 200ul of m-wash buffer to the column and centrifuge at 14000rpm for 30sec
- Add 10ul of EBuffer to column sit for 5 mintues and centrifuge at 14000rpm for 30sec (2 rxn total)
ssDNA Qubit assay[edit]
- 47.1ng/ul *20ul = 942ng
- Stored in Maytag -20C (Alan Misc. #9)
Probe calculations[edit]
illumina methylation seq pool 155[edit]
ppMALAT1[edit]
Capture setup[edit]
GP1_V6
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Sample
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Conc. (ng/ul)
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Volume for 200ng (ul)
|
H2O
|
GP1_V6
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10X ampligase Buffer
|
Total
|
CT Jurkat
|
47.1
|
4.25
|
11.47
|
2.28
|
2
|
20
|
|
|
|
|
|
|
|
illumina methylation seq pool
|
|
|
|
|
|
|
Sample
|
Conc. (ng/ul)
|
Volume for 200ng (ul)
|
H2O
|
illumina methylation seq pool
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10X ampligase Buffer
|
Total
|
CT Jurkat
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47.1
|
4.25
|
10.38
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3.37
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2.00
|
20
|
|
|
|
|
|
|
|
ppMALAT1
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|
|
|
|
|
|
Sample
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Conc. (ng/ul)
|
Volume for 200ng (ul)
|
H2O
|
ppMALAT1
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10X ampligase Buffer
|
Total
|
Jurkat
|
100
|
2
|
15.34
|
0.66
|
2.00
|
20
|
Negative Control
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n/a
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n/a
|
17.34
|
0.66
|
2.00
|
20
|
illumina methylation seq pool 155 probes setup[edit]
Program stored under Alan/aCPG
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 58C 20h
-> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
GP1_V6 & ppMALAT1 setup[edit]
Program stored under Kun/CPG
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h
-> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
- GP1_V6-Jurkat Stored in Maytag -20C (Alan Misc. #24)
- illumina_155-Jurkat Stored in Maytag -20C (Alan Misc. #25, remove for amplification done on 01282014)
- ppMALTA1-Jurkat Stored in Maytag -20C (Alan Misc. #26)
- ppMALTA1-H2O Stored in Maytag -20C (Alan Misc. #27)
Prepare SLN Mix[edit]
SLN Mix
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Components
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Stock Concentration
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Unit
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Final Concentration
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Unit
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Prepare Volume (ul)
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AmpliTaq Stoffel
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10
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U/ul
|
2
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U/ul
|
2.6
|
Ampligase
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5
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U/ul
|
0.5
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U/ul
|
1.3
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dNTP
|
1
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mM
|
100
|
uM
|
1.3
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10X Ampligase Buffer
|
10
|
X
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1
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X
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1.3
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H2O
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n/a
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n/a
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n/a
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n/a
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6.5
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Total
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n/a
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n/a
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n/a
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n/a
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13
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Prepare Exo Mix[edit]
- Mix 1:1 Exo I & Exo III (9ul total)
PCR Amplification[edit]
(AmpF6.4Sol and AmpR6.3 for GP1_V6 & illumina_155)[edit]
(ISB_CA_AF and ISB_CA_AR_T2 for ppMALTA1) stored in #18, #19 Alan Misc. -20C[edit]
Probes
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BSPP_GP1_V6
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illumina_155
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Sample
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Jurkat_CT
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Jurkat_CT
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Index
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1
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2
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Components
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1x rxn
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1x rxn
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Captured Template
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20
|
20
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10uM AmpF6.4Sol
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1
|
1
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10uM AmpR6.3Indx
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1
|
1
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2X KAPA SYBR MM
|
25
|
25
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H2O
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3
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3
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|
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Probes
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ppMALTA1
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ppMALTA1
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Sample
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Jurkat_gDNA
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-ve control
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Components
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1x rxn
|
1x rxn
|
Captured Template
|
20
|
20
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10uM ISB_CA_AF
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1
|
1
|
10uM ISB_CA_AR_T2
|
1
|
1
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2X KAPA SYBR MM
|
25
|
25
|
H2O
|
3
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3
|
Program (Eppendorf Realplex stored under Alan/ampF6.4_template)
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
File:01212014 qpcr 1.JPG
- After the 8+15 cycle only GP1_V6 captured seq library got amplified
- After meeting with Dr. Zhang, he suggests adding more cycles to the remaining 3 captured product
File:01212014 qpcr.JPG
- Amplification happened immediately for all 3 captures, that includes the negative control, which makes me think it is unspecific amplification. I will run a PAGE gel to verify the amplicon size
- GP1_V6 amplicon Stored in Maytag -20C (Alan Misc. #20)
- illumina_155 amplicon Stored in Maytag -20C (Alan Misc. #21, removed for gel size selection)
- ppMALTA1_Jurkat amplicon Stored in Maytag -20C (Alan Misc. #22)
- ppMALTA1_H2O amplicon Stored in Maytag -20C (Alan Misc. #23)
PAGE Gel[edit]
File:ZhangLab 2 2014-01-21 13hr 54min.jpg
- LM-GP1_V6-illumina_155-ppMALAT1-ppMALAT1_Negative
- illumina_155: probe:99nt, gap:99nt, primer: Dr. Zhang agrees that the amplicon at around 300bp should be the captured product. We will perform a size selection and send it to illumina for miseq run
Perform PAGE Size Selection on illumina_155pp-CT_Jurkat[edit]
- Purify amplicon using minelute, elute with 12ul EB buffer
- nanodrop result 37.9ng/ul * 12ul = 454.8ng/ul
- PAGE
Re-amplify illumina_JurkatCT 01/28/2014[edit]
Probes
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illumina_155
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Sample
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Jurkat_CT
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Index
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1
|
Captured Template
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4
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10uM AmpF6.4Sol
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1
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10uM AmpR6.3Ind1
|
1
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2X KAPA SYBR MM
|
25
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H2O
|
19
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Total
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50
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Program (Eppendorf Realplex stored under Alan/ampF6.4_template)
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x30 -> 72C 3min
File:01282014.JPG
- Minelute with 15ul EB Buffer
- 14.3ng/ul *15ul = 214.5ng
PAGE Size Selection[edit]
- Lowmass-Ladder: 1ul + 12ul h2o + 13ul loading dye
- Library:13ul library + 13ul 6x loading dye
- One well in a 5 well TBE gel, run at 200V for 30 mins
File:ZhangLab 2 2014-01-28 15hr 26min.jpg
HL-BSPP-illumina_155pp_CTJurkat-Jan14 Sequencing at illumina[edit]
Prepare sequencing primer[edit]
Read
|
Primer
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Read 1
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SolSeq6.3.3
|
Index Read
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AmpR6.3IndSeq
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Read 2
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SolSeqV6.3.2r
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- Spike in (100uM) primer to Miseq cartridge (1mL)
|
Stock Primer 100uM
|
Spike into Total Volume (ul)
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Final Concentration (uM)
|
Volume (ul)
|
5
|
1000
|
0.5
|
Prepare Sequencing Library[edit]
- Library concentration 1.69nM (qPCR), we normally load it at 10pM