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= BSPP capture of GP1 samples = * I have helped Alan to continue GP1 BSPP preparation and BSPP capture on cancer cell and blood samples. * [http://genome-tech.ucsd.edu/LabNotes/index.php/AlanFung:LabNotes/Probe/2013-12-11| Alan's note] * Alan performed 24 PCR reaction for production PCR for 15 cycles and I continued EtOH precipitation and column re-purification. * EtOH precipitation of the 24 rxn PCR in2x 15mL tube ** 1200ul amplified amplicons + 120ul 3M NaOAc pH5.5 + 4ul Glycoblue and 3mL 100% EtOH ** Incubate at -80C ~5hr --> * Resuspend each tube with 100ul H2O, purified with 2X QIAGEN column, eluted 50ul each tube. After ND measurement, total volume for each set is ~90ul) ** V4 set: conc. dsDNA (ND) = 54.7ng/ul, total yield = 90ul * 54.7ng/ul = 4.92ug ** V6 set: conc. dsDNA (ND) = 68.4ng/ul, total yield = 90ul * 68.4ng/ul = 6.16ug ** Note. The total yield was expected to be ~25+/-5ug for 24 reactions, but I got only XX ug. This is possible that, Alan amplified only 9 cycles in expansion PCR. Since >90% of seed oligos from CustomAraays are inclomplete oligoes. There could be more fraction of these incomplete oligos in the first amplicons as we used very high template concentration and amplified with fewer cycles (I always amplified 15 cycles for expansion PCR and production PCR consistently.) == Lambda Exonuclease digestion == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' |- | ds amplicons ||90.00 |- | 10X Lambda Exo buffer||10.50 |- | Lambda Exo (5U/ul)||5.00 |- | Total||105.50 |} * Incubate at 37C for 1hr * Purified with 1X QIAQuick column for each set (I added 7x vol of PB buffer = 770ul. Total volume is 875ul, so I add 750ul of solution mix -> spin and add the rest to the column again.) * Eluted with 40ul EB buffer each (total volume left after ND measurement ~37.5ul) ** V4 set: conc. ssDNA (ND) = 34.6ng/ul, yield = 37.5ul * 34.6ng/ul = 1.3ug ** V6 set: conc. ssDNA (ND) = 49.7ng/ul, yield = 37.5ul * 49.7ng/ul = 1.86ug == Removal of amplification adapters == * Set up the same volume for both V4 & V6 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' |- | ssDNA ||37.50 |- | USER||2.50 |- | Total||40.00 |} * 1.30ug of V4 in total 40ul & 1.86ug of V6 in total 40ul * Incubate at 37C for 3hr * Add 2.5ul of RE-DpnII-V4 guide oligo to GP1_V4 and 2.5ul of RE-DpnII-V6 guide oligo to GP1_V6. Mix well and spin down. * Incubated at 94 C for 2 min, cool down temperature to 37C for 3 min * Add 2.5ul of DpnII to each tube. Mix well by pipetting up and down 10x while the tube on thermocycler. * Incubate at 37C for 2hr * Heat inactivate at 75C for 20min * Purify with 1x QIAquick column for each set ** I add 700ul of PB buffer, just add extra volume of buffer to fill up the column) * Elute with 60ul EB buffer for each set * Measure concentration with ND (ssDNA, After elution and ND measurement there is ~58ul left total) ** GP1V4 set: conc. ssDNA (ND) = 13.1ng/ul, total yield = 58ul * 13.1ng/ul = 760ng ** GP1V6 set: conc. ssDNA (ND) = 18.9ng/ul, total yield = 58ul * 18.9ng/ul = 1,096ng GP1_V4 GP1_V6 * Perform PAGE size-selection (1x 2D well for each set) [[File:ZhangLab_2 2013-12-22 17hr 34min_GP1_V4V6_PAGE-SS.jpg|600px]] ** Resuspend with 900ul 1X TE buffer in 2mL LoBind tube ** After incubation for 1hr, pass through 2x nanosep and split ~400 to 2x tube for each set. ** Precipitate with 1.34ul glycoblue, 40ul 3M NaOAc pH 5.5, and 1mL 100% EtOH ** Incubate at -80C O/N ** Spin down and resuspend 11ul each tube (total 22ul for each set) * Measure concentration with Qubit ssDNA quantification (1ul for the assay) ** GP1V4 set: conc. ssDNA (ND) = 4.54ng/ul, total yield = 20ul * 4.54ng/ul = 90.8ng ** GP1V6 set: conc. ssDNA (ND) = 7.96ng/ul, total yield = 20ul * 7.96ng/ul = 159.2ng * Verify in 6% TBU gel (load 0.5ul) [[File:ZhangLab_2 2013-12-23 16hr 30min_PG1-gelVerification.jpg| 400px]] ---- = 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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