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= DNA methylation assay - MONOD Project, MONOD V3 12k probe set (BSPP capture) = [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014 Link to calendar]]<br> '''Sample list'''<br> : Sample list will be the same as experiment in 2013-03-02<br> : - Cancer cell lines (5 samples) : - Differentiated H1 & H9 (BMP4 treatment, I also included H1 control, 3 samples) : - Blood samples (3 samples) * I wanted to compare Hemo KlenTaq and Stoffel Fragment, so I will include one of cancer sample with Stoffel Fragment. ==== Probe preparation ==== [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-3-26]] ==== Sample IDs tracking ==== * [http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-3-2#Sample_IDs_tracking Refer to previous experiment on 2014-03-02] == Bisulfite conversion == * * I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo (lot Mar 2014) and followed the protocol of the kit === Bisulfite conversion set up === {| {{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)''' | align="center" style="background:#f0f0f0;"|'''Samples''' |- | CANC_1||47.70||12.58||MN_1||7.42||130.00||150.00||CANC_1 |- | CANC_2||45.40||13.22||MN_2||6.78||130.00||150.00||CANC_2 |- | CANC_3||41.40||14.49||MN_3||5.51||130.00||150.00||CANC_3 |- | CANC_4||47.10||12.74||MN_4||7.26||130.00||150.00||CANC_4 |- | CANC_5||68.20||8.80||MN_5||11.20||130.00||150.00||CANC_5 |- | SR-3||62.20||9.65||MN_6||10.35||130.00||150.00||SR-3 |- | MA-16||85.00||7.06||MN_7||12.94||130.00||150.00||MA-16 |- | MA-17||49.40||12.15||MN_8||7.85||130.00||150.00||MA-17 |- | F10||106.00||5.66||MN_9||14.34||130.00||150.00||F10 |- | G10||108.00||5.56||MN_10||14.44||130.00||150.00||G10 |- | H11||102.00||5.88||MN_11||14.12||130.00||150.00||G11 |- | NTC_HemoKlenTaq||0||0||MN_12||20.00||130.00||150.00||NTC |- | CANC_1||47.70||12.58||MN_13||7.42||130.00||150.00||CANC_1 |- | NTC_Stoffel||0||0||MN_14||20.00||130.00||150.00||NTC |} : - Sample G11 was not enough, so I changed to smaple H11. '''Program''' 1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold * I elute with Elution buffer 15ul * I used 1ul for ssDNA Qubit assay. The volume left after assay ~13ul. ==== Qubit ss DNA assay ==== * Dilute 200X (add 1ul of bis-cvt DNA in total 200 assay volume {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''uL used''' | align="center" style="background:#f0f0f0;"|'''Dilution''' | align="center" style="background:#f0f0f0;"|'''Sample conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 13ul (ng)''' | align="center" style="background:#f0f0f0;"|'''% recovery''' |- | MN_1||159||ng/mL||1||200||31.90||414.70||69.12 |- | MN_2||145||ng/mL||1||200||29.10||378.30||63.05 |- | MN_3||113||ng/mL||1||200||22.50||292.50||48.75 |- | MN_4||107||ng/mL||1||200||21.40||278.20||46.37 |- | MN_5||116||ng/mL||1||200||23.10||300.30||50.05 |- | MN_6||138||ng/mL||1||200||27.60||358.80||59.80 |- | MN_7||126||ng/mL||1||200||25.10||326.30||54.38 |- | MN_8||125||ng/mL||1||200||25.00||325.00||54.17 |- | MN_9||101||ng/mL||1||200||20.20||262.60||43.77 |- | MN_10||252||ng/mL||1||200||50.50||656.50||109.42* |- | MN_11||141||ng/mL||1||200||28.10||365.30||60.88 |- | MN_13||160||ng/mL||1||200||32.00||416.00||69.33 |} : - * This sample always has too high recovery rate. This is consistent with previous experiment. I will use double amount for capture since there might be some contaminant causing the background. == BSPP capture set up == === Probe calculation === '''MONOD_V3_12k, size 12,000'''<br> :{| {{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)||12,000||probes|| |- | Human bis-cvt gDNA required||250||ng|| |- | Human gDNA MW||1.822E+12||g/mole||3.0E+09 * 607.4Da/bp |- | Human gDNA( 200ng)||1.372E-19||mole||250E-09g / 1.822E+12g/mole |- | Probe required (500:1)||6.860E-17||mole||500 * 1.098E-19mole |- | Probe mix MW (size12,000probes, 110nt)||4.018E+08||g/mole||12,000 probes * (110bp *303.7Da/bp+79Da) |- | Mass of probe required||2.757E-08||g||6.860E-17mole * 4.018E+08g/mole |- | Mass of probe required||27.57||ng|| |- | Probe conc||12.2||ng/ul|| |- | Volume of probe||2.26||ul|| |} : - I increased bis-DNA to 250ng. ==== Probe & AmpLigase Buffer mix ==== :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''14.5x''' |- | 10X AmpLigase Buffer||2.00||29.00 |- | MONOD_V3_12k (12.2 ng/ul)||2.26||32.77 |- | Toal ||4.26|| |} {| {{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 250ng (ul)''' | align="center" style="background:#f0f0f0;"|'''Bis-cvt samples''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''MONOD probe (61.20ng)''' | align="center" style="background:#f0f0f0;"|'''10X AmpLigase Buffer''' | align="center" style="background:#f0f0f0;"|'''Total (ul)''' | align="center" style="background:#f0f0f0;"|'''Converted DNA amount (ng)''' |- | MN_1||31.90||7.84||MN_1||7.90||2.26||2.00||20.00||250.00 |- | MN_2||29.10||8.59||MN_2||7.15||2.26||2.00||20.00||250.00 |- | MN_3||22.50||11.11||MN_3||4.63||2.26||2.00||20.00||250.00 |- | MN_4||21.40||11.68||MN_4||4.06||2.26||2.00||20.00||250.00 |- | MN_5||23.10||10.82||MN_5||4.92||2.26||2.00||20.00||250.00 |- | MN_6||27.60||9.06||MN_6||6.68||2.26||2.00||20.00||250.00 |- | MN_7||25.10||9.96||MN_7||5.78||2.26||2.00||20.00||250.00 |- | MN_8||25.00||10.00||MN_8||5.74||2.26||2.00||20.00||250.00 |- | MN_9||20.20||12.38||MN_9||3.36||2.26||2.00||20.00||250.00 |- | MN_10||50.50||9.90||MN_10||5.84||2.26||2.00||20.00||499.95** |- | MN_11||28.10||8.90||MN_11||6.84||2.26||2.00||20.00||250.00 |- | MN_13||32.00||7.81||MN_13||7.93||2.26||2.00||20.00||250.00 |- | NTC_HemoKlentaq||0.00||0.00||NTC||15.74||2.26||2.00||20.00||0.00 |- | NTC_Stoffel||0.00||0.00||NTC_Stoffel||15.74||2.26||2.00||20.00||0.00 |} '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2.5ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 200uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. ==== SLN mix solution (Hemo KlenTaq) ==== :{| {{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 125ul''' |- | |||||||||| |- | Hemo Klentaq||10||U/ul||2||U/ul||25.00 |- | AmpLigase||5||U/ul||0.5||U/ul||12.50 |- | dNTP||bgcolor="lawngreen"|10||mM||200||uM||2.50 |- | 10x AmpLigase Buffer||10||x||1||x||12.50 |- | H2O||||||||||72.50 |} : - I prepare extra for MONOD_GP1_V4 capture experiment ==== SLN mix solution (Stoffel Fragment) ==== :{| {{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 10ul''' |- | |||||||||| |- | Stoffel Fragment||10||U/ul||2||U/ul||2.00 |- | AmpLigase||5||U/ul||0.5||U/ul||1.00 |- | dNTP||1||mM||200||uM||2.00 |- | 10x AmpLigase Buffer||10||x||1||x||1.00 |- | H2O||||||||||4.00 |} == Amplification == : - Add 20% v/v of captured reaction in 100ul PCR (KAPA) '''Program (Eppendorf Realplex)''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min : - Sample MN13 stopped at 20 cycles : - Sample MN1,3, 6 stopped at 21 cycles : - Samples MN2,4,5,7-11 stopped at 23 cycles : - Verify 4ul of PCR product in 6% TBE gel [[File:ZhangLab_2 2014-04-02 13hr 46min_MONOD_v3capture_gel1.jpg| 400px]] [[File:ZhangLab_2 2014-04-02 13hr 46min_MONOD_v3capture_gel2.jpg|325px]] : - Purify PCR product with 0.7 volume of AMPure beads : - Eluted with 60ul H2O : - Quantify concentration in 6% TBE gel (load 2ul of purified libraries) [[File:ZhangLab_2 2014-04-02 17hr 21min_MONOD_V3_Seq_PQ_1-8.jpg| 400px]] [[File:ZhangLab_2 2014-04-02 17hr 21min_MONOD_V3_Seq_PQ_9-13.jpg| 250px]] {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Original IDs''' | align="center" style="background:#f0f0f0;"|'''Index''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount in 58ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 50ng (ul)''' |- | MN_1||BXPC3||Ind_1||1.41||81.93||35.39 |- | MN_2||U87MG||Ind_2||1.12||64.93||44.67 |- | MN_3||PANC1||Ind_3||1.67||97.11||29.86 |- | MN_4||BE(2)C||Ind_4||3.13||181.61||15.97 |- | MN_5||T98G||Ind_5||3.74||216.67||13.38 |- | MN_6||H9 p43 BMP4||Ind_6||2.13||123.51||23.48 |- | MN_7||H1 p47 Control||Ind_7||2.96||171.40||16.92 |- | MN_8||H1 p47 BMP-4||Ind_8||2.86||165.82||17.49 |- | MN_9||UCLA-B-F10||Ind_9||3.27||189.49||15.30 |- | MN_10||UCLA-B-G10||Ind_10||2.75||159.39||18.19 |- | MN_11||UCLA-B-H11||Ind_11||0.91||52.65||55.08 |- | MN_13||BXPC3_Stoffel||Ind_12||2.68||155.23||18.68 |} : - Pool equal amount of sequencing libraries (50ng each) and performed PAGE size-selection in 2 of 5-well 6% TBE gels at the band ~375bp [[File:ZhangLab_2 2014-04-09 11hr 37min_MONOD_V3_SeqLib_PAGESS-1.jpg| 230px]] [[File:ZhangLab_2 2014-04-09 11hr 37min_MONOD_V3_SeqLib_PAGESS-2.jpg| 240px]] : - Elute with 30ul H2O : - Verify sequencing libraries in 6% TBE gel by loading 2ul '''Sequencing ibrary IDs: NP-BSPP-MONOD_V3_Mar30, 2014'''<br> '''Sequencing run: PE run, 150+7+150 bp''' : - MiSeq run: 140411_MiSeq (PE 76 + 6 + 76 in stead of PE 150+7+150 bp)
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