AlanFung:LabNotes/Capturing/2014-1-14

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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]

GP1_V6[edit]

Capture setup[edit]

GP1_V6            
Sample Conc. (ng/ul) Volume for 200ng (ul) H2O GP1_V6 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 10X ampligase Buffer Total
CT Jurkat 47.1 4.25 10.38 3.37 2.00 20
         
ppMALAT1            
Sample Conc. (ng/ul) Volume for 200ng (ul) H2O ppMALAT1 10X ampligase Buffer Total
Jurkat 100 2 15.34 0.66 2.00 20
Negative Control n/a 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          
           
Components Stock Concentration Unit Final Concentration Unit Prepare Volume (ul)
AmpliTaq Stoffel 10 U/ul 2 U/ul 2.6
Ampligase 5 U/ul 0.5 U/ul 1.3
dNTP 1 mM 100 uM 1.3
10X Ampligase Buffer 10 X 1 X 1.3
H2O n/a n/a n/a n/a 6.5
Total n/a n/a n/a n/a 13

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 BSPP_GP1_V6 illumina_155
Sample Jurkat_CT Jurkat_CT
Index 1 2
Components 1x rxn 1x rxn
Captured Template 20 20
10uM AmpF6.4Sol 1 1
10uM AmpR6.3Indx 1 1
2X KAPA SYBR MM 25 25
H2O 3 3
     
Probes ppMALTA1 ppMALTA1
Sample Jurkat_gDNA -ve control
Components 1x rxn 1x rxn
Captured Template 20 20
10uM ISB_CA_AF 1 1
10uM ISB_CA_AR_T2 1 1
2X KAPA SYBR MM 25 25
H2O 3 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 illumina_155
Sample Jurkat_CT
Index 1
Captured Template 4
10uM AmpF6.4Sol 1
10uM AmpR6.3Ind1 1
2X KAPA SYBR MM 25
H2O 19
Total 50
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
Read 1 SolSeq6.3.3
Index Read AmpR6.3IndSeq
Read 2 SolSeqV6.3.2r
  • Spike in (100uM) primer to Miseq cartridge (1mL)
  Stock Primer 100uM Spike into Total Volume (ul) Final Concentration (uM)
Volume (ul) 5 1000 0.5

Prepare Sequencing Library[edit]

  • Library concentration 1.69nM (qPCR), we normally load it at 10pM