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= DNA methylation assay - MONOD Project (BSPP capture) = [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014 Link to calendar]]<br> '''Sample list''' : - Cancer cell lines (5 samples) : - Differentiated H1 & H9 (BMP4 treatment, I also included H1 control, 3 samples) : - Blood samples (3 samples) ==== Sample IDs tracking ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Original ID''' |- | CANC_1||BXPC3 |- | CANC_2||U87MG |- | CANC_3||PANC1 |- | CANC_4||BE(2)C |- | CANC_5||T98G |- | SR-3||H9 p43 BMP4 |- | MA-16||H1 p47 Control |- | MA-17||H1 p47 BMP-4 |- | F10||UCLA-B-F10 |- | G10||UCLA-B-G10 |- | G11||UCLA-B-G11 |} == Bisulfite conversion == * I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo 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''' |- | CANC_1||47.70||12.58||MN#1||7.42||130.00 |- | CANC_2||45.40||13.22||MN#2||6.78||130.00 |- | CANC_3||41.40||14.49||MN#3||5.51||130.00 |- | CANC_4||47.10||12.74||MN#4||7.26||130.00 |- | CANC_5||68.20||8.80||MN#5||11.20||130.00 |- | SR-3||62.20||9.65||MN#6||10.35||130.00 |- | MA-16||85.00||7.06||MN#7||12.94||130.00 |- | MA-17||49.40||12.15||MN#8||7.85||130.00 |- | F10||106.00||5.66||MN#9||14.34||130.00 |- | G10||108.00||5.56||MN#10||14.44||130.00 |- | G11||82.60||7.26||MN#11||12.74||130.00 |- | NTC||0||0||MN#12||20.00||130.00 |} '''Program''' 1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold * I elute with Elution buffer 16ul * 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;"|'''''' | align="center" style="background:#f0f0f0;"|'''uL used''' | align="center" style="background:#f0f0f0;"|'''Dilution''' | align="center" style="background:#f0f0f0;"|'''Sample conc.''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Yields in 13ul (ng)''' | align="center" style="background:#f0f0f0;"|'''% recovery''' |- | MN_1||175||ng/mL||1||200||34.9||ng/ul||453.7||75.62 |- | MN_2||122||ng/mL||1||200||24.5||ug/mL||318.5||53.08 |- | MN_3||122||ng/mL||1||200||24.5||ug/mL||318.5||53.08 |- | MN_4||87.6||ng/mL||1||200||17.5||ug/mL||227.5||37.92 |- | MN_5||108||ng/mL||1||200||21.7||ug/mL||282.1||47.02 |- | MN_6||131||ng/mL||1||200||26.2||ug/mL||340.6||56.77 |- | MN_7||107||ng/mL||1||200||21.4||ug/mL||278.2||46.37 |- | MN_8||71.4||ng/mL||1||200||14.3||ug/mL||185.9||30.98 |- | MN_9||61.5||ng/mL||1||200||12.3||ug/mL||159.9||26.65 |- | MN_10||173||ng/mL||1||200||34.6||ug/mL||449.8||74.97 |- | MN_11||72.9||ng/mL||1||200||14.6||ug/mL||189.8||31.63 |} == BSPP capture set up == === Probe calculation === {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe:taeget''' | align="center" style="background:#f0f0f0;"|'''200:1''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Calculation''' |- | Probe size (number of individual probe in oligo mix)||83447||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 (200:1)||2.195E-17||mole||200 * 1.098E-19mole |- | Probe mix MW (size 83447probes, 110nt)||2.788E+09||g/mole||83447 probes * 110bp *303.7Da/bp |- | Mass of probe required||6.119E-08||g||2.195E-17mole * 2.788E+09g/mole |- | Mass of probe required||61.20||ng|| |- | Probe conc||11.9||ng/ul|| |- | Volume of probe||5.14||ul|| |} ==== Probe & AmpLigase Buffer mix ==== :{| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''12.5x''' |- | 10X AmpLigase Buffer||2.00||25.00 |- | MONOD (11.90ng/ul)||5.14||64.25 |- | Toal ||7.14|| |} {| {{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;"|'''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||34.90||5.73||MN_1||7.13||5.14||2.00||20.00||200.00 |- | MN_2||24.50||8.16||MN_2||4.70||5.14||2.00||20.00||200.00 |- | MN_3||24.50||8.16||MN_3||4.70||5.14||2.00||20.00||200.00 |- | MN_4||17.50||11.43||MN_4||1.43||5.14||2.00||20.00||200.00 |- | MN_5||21.70||9.22||MN_5||3.64||5.14||2.00||20.00||200.00 |- | MN_6||26.20||7.63||MN_6||5.23||5.14||2.00||20.00||200.00 |- | MN_7||21.40||9.35||MN_7||3.51||5.14||2.00||20.00||200.00 |- | MN_8||14.30||13.00||MN_8||0.00||5.14||2.00||20.14||185.90 |- | MN_9||12.30||13.00||MN_9||0.00||5.14||2.00||20.14||159.90 |- | MN_10||34.60||5.78||MN_10||7.08||5.14||2.00||20.00||200.00 |- | MN_11||14.60||13.00||MN_11||0.00||5.14||2.00||20.14||189.80 |- | MN_12||0.00||0.00||MN_12||12.86||5.14||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; 100uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. * Note: Concentration of Hemo Klentaq was not provided, so I assumed it has the same concentration as Stoffel Fragment ==== SLN mix solution ==== :{| {{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)<br>total 40ul''' |- | Hemo Klentaq||10||U/ul||2||U/ul||8.00 |- | AmpLigase||5||U/ul||0.5||U/ul||4.00 |- | dNTP||1||mM||100||uM||4.00 |- | 10x AmpLigase Buffer||10||x||1||x||4.00 |- | H2O||||||||||20.00 |} ==== 2014-03-05==== == PCR Amplification (TEST) == {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''12.5x rxn mix''' |- | Captured template||4.00||0.00 |- | 10uM AmpF6.4Sol||0.50||6.25 |- | 10uM AmpR6.3Indx1||0.50||6.25 |- | 2X KAPA SYBR MM||12.50||156.25 |- | H2O||7.50||93.75 |- | Total||25.00|| |} - Aliquot 21ul, add 4ul of circularized DNA<br> '''Program (Eppendorf Realplex)''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min ==== qPCR curves ==== [[File:2014-03-05_qPCR_TEST_MONOD_capture.png| 600px]] ==== Verified amplified products in 6% TBE gel ==== * Loaded 3ul of PCR product in 6% TBE gel [[File:ZhangLab_2 2014-03-05 13hr 23min_TEST_PCR_MONOD_MN_1-8.jpg|450px]] [[File:ZhangLab_2 2014-03-05 13hr 23min_TEST_PCR_MONOD_MN_9-11.jpg|260px]] * If everything works well, I will set up duplicate reactions of 50ul with 20% (v/v) of template {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''25x rxn mix''' |- | Captured template||10.00||0.00 |- | 10uM AmpF6.4Sol||1.00||25.00 |- | 10uM AmpR6.3IndX(X=81-92)||1.00||0.00 |- | 2X KAPA SYBR MM||25.00||625.00 |- | H2O||13.00||325.00 |- | Total||50.00|| |} - Aliquot 39ul, add 1ul of 10uM AmpR6.3IndX, add 10ul of circularized DNA<br> '''Program (Eppendorf Realplex)''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min ==== qPCR curves ==== I took the average of the replicates [[File:2014-03-05_qPCR_MONOD_capture.png| 600px]] : - Stopped MN_1-8 at 14 cycles : - Stopped MN_9-11 at 18 cycles : - Pool and purify PCR product with 0.7 volume of AMPure beads : - Eluted with 65ul H2O : - Quantify concentration in 6% TBE gel (load 1.5ul of purified libraries) :[[File:ZhangLab_2 2014-03-05 17hr 20min_MONOD_SeqLib_PQind.jpg|450px]] {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Original IDs''' | align="center" style="background:#f0f0f0;"|'''Indexes''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount in 65ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 50ng (ul)''' |- | MN#1||BXPC3||Ind81||2.16||140.17||23.19 |- | MN#2||U87MG||Ind82||3.14||204.13||15.92 |- | MN#3||PANC1||Ind83||6.28||408.34||7.96 |- | MN#4||BE(2)C||Ind84||3.76||244.61||13.29 |- | MN#5||T98G||Ind85||1.90||123.48||26.32 |- | MN#6||H9 p43 BMP4||Ind86||8.26||536.74||6.06 |- | MN#7||H1 p47 Control||Ind87||2.77||180.10||18.05 |- | MN#8||H1 p47 BMP-4||Ind88||1.08||70.34||46.20 |- | MN#9||UCLA-B-F10||Ind89||3.32||216.00||15.05 |- | MN#10||UCLA-B-G10||Ind90||5.65||367.18||8.85 |- | MN#11||UCLA-B-G11||Ind91||1.50||97.31||33.40 |} : - Pool equal amount of sequencing libraries and performed PAGE size-selection in 2 of 5-well 6% TBE gels at the band ~375bp : [[File:ZhangLab_2 2014-03-05 18hr 59min_PAGE-SS.jpg| 500px]] : - Elute with 24ul H2O. : - Verify sequencing libraries in 6% TBE gel by loading 2ul : [[File:ZhangLab_2 2014-03-06 18hr 21min_verify_MONOD-SeqLib.jpg| 100px]] : -Hand to Alan for concentration quantification by qPCR '''Sequencing ibrary IDs: NP-BSPP-MONOD_81-91_Mar2, 2014'''<br> '''Sequencing run: ''' Dr. Zhang suggested to do MiSeq PE run, 150+7+150 bp : - MiSeq run: '''MONOD_81_91''' on March 3rd, 2014'''
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