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==Capturing Bisulfite Converted Jurkat gDNA w GP1 V6 probe set== *probes prep notes LabNotes/Probe/2014-1-7 [[http://genome-tech.ucsd.edu/LabNotes/index.php/AlanFung:LabNotes/Probe/2014-1-7#ETOH_Precipitation]] ==Capturing Jurkat gDNA w ppMALT1 ordered form IDT== ==Bisulfite Conversion of Jurkat gDNA== *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== *47.1ng/ul *20ul = 942ng *Stored in Maytag -20C (Alan Misc. #9) ==Probe calculations== ===illumina methylation seq pool 155=== ===ppMALAT1=== ===GP1_V6=== ==Capture setup== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- align="center" | width="157" height="13" | GP1_V6 | align="center" width="75" | | align="center" width="120" | | align="center" width="75" | | align="center" width="157" | | align="center" width="116" | | align="center" width="75" | |- align="center" | height="13" | Sample | Conc. (ng/ul) | Volume for 200ng (ul) | H2O | GP1_V6 | 10X ampligase Buffer | Total |- align="center" | height="13" | CT Jurkat | align="center" | 47.1 | align="center" | 4.25 | align="center" | 11.47 | align="center" | 2.28 | align="center" | 2 | align="center" | 20 |- align="center" | align="center" height="13" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | |- align="center" | height="13" | illumina methylation seq pool | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | |- align="center" | height="13" | Sample | Conc. (ng/ul) | Volume for 200ng (ul) | H2O | illumina methylation seq pool | 10X ampligase Buffer | Total |- align="center" | height="13" | CT Jurkat | align="center" | 47.1 | align="center" | 4.25 | align="center" | 10.38 | align="center" | 3.37 | align="center" | 2.00 | align="center" | 20 |- align="center" | align="center" height="13" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | |- align="center" | height="13" | ppMALAT1 | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | |- align="center" | height="13" | Sample | Conc. (ng/ul) | Volume for 200ng (ul) | H2O | ppMALAT1 | 10X ampligase Buffer | Total |- align="center" | height="13" | Jurkat | align="center" | 100 | align="center" | 2 | align="center" | 15.34 | align="center" | 0.66 | align="center" | 2.00 | align="center" | 20 |- align="center" | height="13" | Negative Control | n/a | n/a | align="center" | 17.34 | align="center" | 0.66 | align="center" | 2.00 | align="center" | 20 |} ===illumina methylation seq pool 155 probes setup=== 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=== 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== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- align="center" | width="117" height="13" | SLN Mix | align="center" width="110" | | align="center" width="75" | | align="center" width="75" | | align="center" width="75" | | align="center" width="109" | |- align="center" | align="center" height="13" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | |- align="center" | height="13" | Components | Stock Concentration | Unit | Final Concentration | Unit | Prepare Volume (ul) |- align="center" | height="13" | AmpliTaq Stoffel | align="center" | 10 | U/ul | align="center" | 2 | U/ul | align="center" | 2.6 |- align="center" | height="13" | Ampligase | align="center" | 5 | U/ul | align="center" | 0.5 | U/ul | align="center" | 1.3 |- align="center" | height="13" | dNTP | align="center" | 1 | mM | align="center" | 100 | uM | align="center" | 1.3 |- align="center" | height="13" | 10X Ampligase Buffer | align="center" | 10 | X | align="center" | 1 | X | align="center" | 1.3 |- align="center" | height="13" | H2O | n/a | n/a | n/a | n/a | align="center" | 6.5 |- align="center" | height="13" | Total | n/a | n/a | n/a | n/a | align="center" | 13 |} ==Prepare Exo Mix== *Mix 1:1 Exo I & Exo III (9ul total) ==PCR Amplification== ===(AmpF6.4Sol and AmpR6.3 for GP1_V6 & illumina_155)=== ===(ISB_CA_AF and ISB_CA_AR_T2 for ppMALTA1) stored in #18, #19 Alan Misc. -20C=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="111" height="15" | Probes | width="77" | BSPP_GP1_V6 | width="65" | illumina_155 |- style="font-size:12pt;font-weight:bold" align="center" | height="15" | Sample | Jurkat_CT | Jurkat_CT |- style="font-size:12pt;font-weight:bold" align="center" | height="15" | Index | align="center" | 1 | align="center" | 2 |- style="font-size:12pt" align="center" | height="15" | Components | 1x rxn | 1x rxn |- style="font-size:12pt" align="center" | height="15" | Captured Template | align="center" | 20 | align="center" | 20 |- style="font-size:12pt" align="center" | height="15" | 10uM AmpF6.4Sol | align="center" | 1 | align="center" | 1 |- style="font-size:12pt" align="center" | height="15" | 10uM AmpR6.3Indx | align="center" | 1 | align="center" | 1 |- style="font-size:12pt" align="center" | height="15" | 2X KAPA SYBR MM | align="center" | 25 | align="center" | 25 |- style="font-size:12pt" align="center" | height="15" | H2O | align="center" | 3 | align="center" | 3 |- style="font-size:12pt" align="center" | align="center" height="15" | | align="center" | | align="center" | |- style="font-size:12pt;font-weight:bold" align="center" | height="15" | Probes | ppMALTA1 | ppMALTA1 |- style="font-size:12pt;font-weight:bold" align="center" | height="15" | Sample | Jurkat_gDNA | -ve control |- style="font-size:12pt" align="center" | height="15" | Components | 1x rxn | 1x rxn |- style="font-size:12pt" align="center" | height="15" | Captured Template | align="center" | 20 | align="center" | 20 |- style="font-size:12pt" align="center" | height="15" | 10uM ISB_CA_AF | align="center" | 1 | align="center" | 1 |- style="font-size:12pt" align="center" | height="15" | 10uM ISB_CA_AR_T2 | align="center" | 1 | align="center" | 1 |- style="font-size:12pt" align="center" | height="15" | 2X KAPA SYBR MM | align="center" | 25 | align="center" | 25 |- style="font-size:12pt" align="center" | height="15" | H2O | align="center" | 3 | align="center" | 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|600px]] *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|600px]] *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== [[File:ZhangLab_2 2014-01-21 13hr 54min.jpg|400px]] *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== *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== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="105" height="15" | Probes | width="71" | illumina_155 |- style="font-size:12pt;font-weight:bold" align="center" | height="15" | Sample | Jurkat_CT |- style="font-size:12pt;font-weight:bold" align="center" | height="15" | Index | align="center" | 1 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Captured Template | align="center" | 4 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 10uM AmpF6.4Sol | align="center" | 1 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 10uM AmpR6.3Ind1 | align="center" | 1 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 2X KAPA SYBR MM | align="center" | 25 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | H2O | align="center" | 19 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Total | align="center" | 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|600px]] *Minelute with 15ul EB Buffer *14.3ng/ul *15ul = 214.5ng ==PAGE Size Selection== *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|400px]] ==HL-BSPP-illumina_155pp_CTJurkat-Jan14 Sequencing at illumina== ===Prepare sequencing primer=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="116" height="15" | Read | width="263" | Primer |- align="center" |style="font-size:12pt;font-weight:bold" height="16" | Read 1 |style="font-size:13pt" | SolSeq6.3.3 |- align="center" |style="font-size:12pt;font-weight:bold" height="16" | Index Read |style="font-size:13pt" | AmpR6.3IndSeq |- align="center" |style="font-size:12pt;font-weight:bold" height="16" | Read 2 |style="font-size:13pt" | SolSeqV6.3.2r |} *Spike in (100uM) primer to Miseq cartridge (1mL) {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt" align="center" | align="center" width="116" height="16" | |style="font-weight:bold" width="263" | Stock Primer 100uM |style="font-weight:bold" width="179" | Spike into Total Volume (ul) |style="font-weight:bold" width="156" | Final Concentration (uM) |- style="font-size:12pt" align="center" |style="font-weight:bold" height="16" | Volume (ul) | align="center" | 5 | align="center" | 1000 | align="center" | 0.5 |} ===Prepare Sequencing Library=== *Library concentration '''1.69nM''' (qPCR), we normally load it at 10pM
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