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= BSPP capture = * Alan extracted gDNA of 5 cancer cell lines. He split the cell into duplicates, so there were two tubes of purified gDNA. * He measured gDNA conc. of cancer cell line and blood sample by Qubit dsDNA assay kit. ==== Qubit result ==== {| {{table}} class="wikitable" style="width: 30%; height: 200px" border="1" | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Sample Concentration (ng/ul)''' |- | BXPC3_1||63.00 |- | BXPC3_2||70.40 |- | U87MG_1||57.20 |- | U87MG_2||63.00 |- | PANC1_1||52.40 |- | PANC1_2||57.80 |- | BE(2)C_1||49.80 |- | BE(2)C_2||50.00 |- | T98G_1||68.20 |- | T98G_2||69.20 |- | B1||102.00 |- | D1||110.00 |- | F10||106.00 |- | G10||108.00 |- | G11||82.60 |- | H11||102.00 |} == Bisulfite conversion == ==== Sample IDs and labeling matching ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''V4 Labeled IDs''' | align="center" style="background:#f0f0f0;"|'''V6 labeled IDs''' |- | BXPC3_1||V4-1||V6-1 |- | U87MG_1||V4-2||V6-2 |- | PANC1_1||V4-3||V6-3 |- | BE(2)C_1||V4-4||V6-4 |- | T98G_1||V4-5||V6-5 |- | B1||V4-6||V6-6 |- | D1||V4-7||V6-7 |- | H11||V4-8||V6-8 |} * I used set _1 of cancel cells and B1, D1 and H11 blood samples for BSPP capture. * I used the new DNA methylation lightning kit (column) ordered by Dr. Zhang in November 2013. * I did bisulfite conversion in duplicates for the two probe sets capture. {| {{table}} class="wikitable" | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 600ng (ul)''' | align="center" style="background:#f0f0f0;"|'''Go to tube''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Lightning Conv Reagent''' | align="center" style="background:#f0f0f0;"|'''Total (ul)''' |- | BXPC3_1||63.00||9.52||A1||10.48||130.00||150.00 |- | U87MG_1||57.20||10.49||A2||9.51||130.00||150.00 |- | PANC1_1||52.40||11.45||A3||8.55||130.00||150.00 |- | BE(2)C_1||49.80||12.05||A4||7.95||130.00||150.00 |- | T98G_1||68.20||8.80||A5||11.20||130.00||150.00 |- | B1||102.00||5.88||A6||14.12||130.00||150.00 |- | D1||110.00||5.45||A7||14.55||130.00||150.00 |- | H11||102.00||5.88||A8||14.12||130.00||150.00 |- | BXPC3_1||63.00||9.52||B1||10.48||130.00||150.00 |- | U87MG_1||57.20||10.49||B2||9.51||130.00||150.00 |- | PANC1_1||52.40||11.45||B3||8.55||130.00||150.00 |- | BE(2)C_1||49.80||12.05||B4||7.95||130.00||150.00 |- | T98G_1||68.20||8.80||B5||11.20||130.00||150.00 |- | B1||102.00||5.88||B6||14.12||130.00||150.00 |- | D1||110.00||5.45||B7||14.55||130.00||150.00 |- | H11||102.00||5.88||B8||14.12||130.00||150.00 |} ''' Program '''<br> 1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold * Eluted bisulfite converted DNA form the bead with 20ul H2O * I used 1ul for ssDNA Qubit assay. The volume left after assay ~16ul ==== ssDNA Qubit assay result ==== {| {{table}} class="wikitable" | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Concentration in the Qubit''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''uL used''' | align="center" style="background:#f0f0f0;"|'''Dilution''' | align="center" style="background:#f0f0f0;"|'''Sample conc. (ng/ul)''' |- | V4-1||63.7||ng/mL||1||200||12.7 |- | V4-2||71.4||ng/mL||1||200||14.3 |- | V4-3||51.4||ng/mL||1||200||10.3 |- | V4-4||30.9||ng/mL||1||200||6.18 |- | V4-5||57.9||ng/mL||1||200||11.6 |- | V4-6||281||ng/mL||1||200||56.2 |- | V4-7||70.1||ng/mL||1||200||14 |- | V4-8||125||ng/mL||1||200||25 |- | V6-1||85.1||ng/mL||1||200||17 |- | V6-2||61.3||ng/mL||1||200||12.3 |- | V6-3||61.3||ng/mL||1||200||12.3 |- | V6-4||70.7||ng/mL||1||200||14.1 |- | V6-5||77.1||ng/mL||1||200||15.4 |- | V6-6||112||ng/mL||1||200||22.5 |- | V6-7||35.6||ng/mL||1||200||7.12 |- | V6-8||100||ng/mL||1||200||20 |} == BSPP capture set up == ==== Probe calculation ==== ====GP1_V4 ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe:taeget''' | align="center" style="background:#f0f0f0;"|'''500:1''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Calculation''' |- | Probe size (number of individual probe in oligo mix)||7110||probes|| |- | Human bis-cvt gDNA required||200||ng|| |- | Human gDNA MW||1.822E+12||g/mole||3.0E+09 * 607.4Da/bp |- | Human gDNA( 200ng)||1.098E-19||mole||200E-09g / 1.822E+12g/mole |- | Probe required (500:1)||5.488E-17||mole||500 * 1.098E-19mole |- | Probe mix MW (size 7110probes, 110nt)||2.375E+08||g/mole||7110 probes * 110bp *303.7Da/bp |- | Mass of probe required||1.304E-08||g||5.448E-17 * 2.375E+08mole |- | Mass of probe required||13||ng|| |- | Probe conc||4.54||ng/ul|| |- | Volume of probe||2.86||ul|| |} * Note: Probe is not enough, so use 2.5ul/sample||= 11.35||ng ====GP1_V6 ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe:taeget''' | align="center" style="background:#f0f0f0;"|'''500:1''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Calculation''' |- | Probe size (number of individual probe in oligo mix)||3662||probes|| |- | Human bis-cvt gDNA required||200||ng|| |- | Human gDNA MW||1.822E+12||g/mole||3.0E+09 * 607.4Da/bp |- | Human gDNA( 200ng)||1.098E-19||mole||200E-09g / 1.822E+12g/mole |- | Probe required (500:1)||5.488E-17||mole||500 * 1.098E-19mole |- | Probe mix MW (size 7110probes, 110nt)||1.223E+08||g/mole||3662 probes * 110bp *303.7Da/bp |- | Mass of probe required||6.714E-09||g||5.448E-17 * 1.223E+08mole |- | Mass of probe required||6.71||ng|| |- | Probe conc||7.96||ng/ul|| |- | Volume of probe||0.84||ul|| |} ==== GP1_V4 set up ==== {| {{table}} class="wikitable" | align="center" style="background:#f0f0f0;"|'''Bis-cvt samples''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 200ng (ul)''' | align="center" style="background:#f0f0f0;"|'''Bis-cvt samples''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''GP1_V4''' | align="center" style="background:#f0f0f0;"|'''10X AmpLigase Buffer''' | align="center" style="background:#f0f0f0;"|'''Total (ul)''' | align="center" style="background:#f0f0f0;"|'''Converted DNA amount''' |- | V4-1||12.70||15.50||V4-1||0.00||2.50||2.00||20.00||196.85 |- | V4-2||14.30||13.99||V4-2||1.51||2.50||2.00||20.00||200.00 |- | V4-3||10.30||15.50||V4-3||0.00||2.50||2.00||20.00||159.65 |- | V4-4||6.18||15.50||V4-4||0.00||2.50||2.00||20.00||95.79 |- | V4-5||11.60||15.50||V4-5||0.00||2.50||2.00||20.00||179.80 |- | V4-6||56.20||7.00||V4-6||8.50||2.50||2.00||20.00||393.40 |- | V4-7||14.00||14.29||V4-7||1.21||2.50||2.00||20.00||200.00 |- | V4-8||25.00||8.00||V4-8||7.50||2.50||2.00||20.00||200.00 |} ==== V4 reaction mix ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''8.5x''' |- | 10X AmpLigase Buffer||2.00||17.00 |- | GP1_V1 (4.54ng/ul)||2.50||21.25 |- | Toal ||4.50|| |} ==== GP1_V6 set up ==== {| {{table}} class="wikitable" | align="center" style="background:#f0f0f0;"|'''Bis-cvt samples''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 200ng (ul)''' | align="center" style="background:#f0f0f0;"|'''Bis-cvt samples''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''GP1_V6''' | align="center" style="background:#f0f0f0;"|'''10X AmpLigase Buffer''' | align="center" style="background:#f0f0f0;"|'''Total (ul)''' | align="center" style="background:#f0f0f0;"|'''Converted DNA amount''' |- | V6-1||17.00||11.76||V6-1||5.40||0.84||2.00||20.00||200.00 |- | V6-2||12.30||16.00||V6-2||1.16||0.84||2.00||20.00||196.80 |- | V6-3||12.30||16.00||V6-3||1.16||0.84||2.00||20.00||196.80 |- | V6-4||14.10||14.18||V6-4||2.98||0.84||2.00||20.00||200.00 |- | V6-5||15.40||12.99||V6-5||4.17||0.84||2.00||20.00||200.00 |- | V6-6||22.50||8.89||V6-6||8.27||0.84||2.00||20.00||200.00 |- | V6-7||7.12||16.00||V6-7||1.16||0.84||2.00||20.00||113.92 |- | V6-8||20.00||10.00||V6-8||7.16||0.84||2.00||20.00||200.00 |} ==== V6 reaction mix ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''8.5x''' |- | 10X AmpLigase Buffer||2.00||17.00 |- | GP1_V1 (7.96ng/ul)||0.84||7.14 |- | Toal ||2.84|| |} '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 16h -> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 6h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. ==== SLN Mix ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Stock conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Prepare volume (ul), total 35ul''' |- | AmpliTaq Stoffel||10||U/ul||2||U/ul||7.00 |- | AmpLigase||5||U/ul||0.5||U/ul||3.50 |- | dNTP||1||mM||100||uM||3.50 |- | 10x AmpLigase Buffer||10||x||1||x||3.50 |- | H2O||||||||||17.50 |} == Amplification == ==== <u>PCR TEST</u> ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''17 x rxn mix''' |- | Captured template||2.50||0.00 |- | 10uM AmpF6.4Sol||0.50||8.50 |- | 10uM AmpR6.3||0.50||8.50 |- | 2X KAPA SYBR MM||12.50||212.50 |- | H2O||9.00||153.00 |- | Total||25.00|| |} :- Aliquot 22.5, add 2.5ul of template ==== <u>PCR with barcoded primers</u> ==== :- Ind1-16, 15% template {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''17 x rxn mix''' |- | Captured template||15.00||0.00 |- | 10uM AmpF6.4Sol||2.00||34.00 |- | 10uM AmpR6.3.IndX (X=1-16)||2.00||0.00 |- | 2X KAPA SYBR MM||50.00||850.00 |- | H2O||31.00||527.00 |- | Total||100.00|| |} :- Aliquot 83, add 2ul of AmpR6.3.IndX, 15ul of template {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Index''' |- | BXPC3_V4||Ind_1 |- | U87MG_V4||Ind_2 |- | PANC1_V4||Ind_3 |- | BE(2)C_V4||Ind_4 |- | T98G_V4||Ind_5 |- | UCLA-SZ_B1_V4||Ind_6 |- | UCLA-SZ_D1_V4||Ind_7 |- | UCLA-SZ_H11_V4||Ind_8 |- | BXPC3_V6||Ind_9 |- | U87MG_V6||Ind_10 |- | PANC1_V6||Ind_11 |- | BE(2)C_V6||Ind_12 |- | T98G_V6||Ind_13 |- | UCLA-SZ_B1_V6||Ind_14 |- | UCLA-SZ_D1_V6||Ind_15 |- | UCLA-SZ_H11_V6||Ind_16 |} ==== PAGE quantification ==== :- Loaded 5ul of each sample (total 60ul after bead purification) in 6% TBE gel '''GP1V4'''<br> [[File:ZhangLab_2 2013-12-25 19hr 44min_GP1_V4_1-8_PQ.jpg| 400px]]<br> '''GP1V6'''<br> [[File:ZhangLab_2 2013-12-25 19hr 46min_GP1_V6_1-8_PQ.jpg|400px]] {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 55ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 30ng''' |- | GP1-V4-1||2.13||116.92||14.11 |- | GP1-V4-2||1.57||86.61||19.05 |- | GP1-V4-3||1.40||77.11||21.40 |- | GP1-V4-4||1.45||79.51||20.75 |- | GP1-V4-5||1.60||88.00||18.75 |- | GP1-V4-6||1.60||88.01||18.75 |- | GP1-V4-7||0.56||30.74||53.67 |- | GP1-V4-8||1.12||61.45||26.85 |} {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' |- | GP1-V4-1||0.190907387||20 |- | GP1-V4-2||N/A||50 |- | GP1-V4-3||N/A||50 |- | GP1-V4-4||N/A||50 |- | GP1-V4-5||N/A||50 |- | GP1-V4-6||0.001140051||40 |- | GP1-V4-7||N/A||50 |- | GP1-V4-8||0.08565092||40 |} ==== PAGE size-selection ==== '''GP1V4'''<br> [[File:ZhangLab_2 2013-12-26 13hr 29min_PAGE-SS_V4.jpg|400px]]<br> '''GP1V6'''<br> [[File:ZhangLab_2 2013-12-26 13hr 42min_PAGE-SS_V6.jpg|400px]] ==== <u>Sequencing library IDs</u> ==== '''GP1V4: NP-GP1V4-Ind1_16-Dec26, 2013'''<br> '''GP1V6: NP-GP1V6-Ind1_16-Dec26, 2013'''<br> Sequence with MiSeq run: '''M01186, PE 151+6+151bp'''
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