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* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] = WGBS library preparation of low input DNA N37 10 tissues samples using KAPA protocol (from Eppendorf) and epMotion = * Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-31 == Preparation of enzyme mix for End repair, A-tailing, Ligation, and Methylation adapters == '''Sample list'''<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Sample code on tubes''' | align="center" style="background:#f0f0f0;"|'''Well IDs''' |- | N37-Cerebellum (CRBL)||N37-1||A1 |- | N37-Colon||N37-2||B1 |- | N37-Frontal lobe (FL)||N37-3||C1 |- | N37-Heart||N37-4||D1 |- | N37-Small intestine (SI)||N37-5||E1 |- | N37-Liver||N37-6||F1 |- | N37-Lung||N37-7||G1 |- | N37-Skeletal muscle (SM)||N37-8||H1 |- | N37-Pancrease||N37-9||A2 |- | N37-Stomach||N37-10||B2 |}<br> === End repair === Sheared gDNA 200ng in 50ul {| {{table}} | align="center" style="background:#f0f0f0;"|'''Total reaction in''' | align="center" style="background:#f0f0f0;"|'''70''' | align="center" style="background:#f0f0f0;"|'''ul''' |- | DNA ||50||ul |- | End repair reaction mix ||20||ul |}<br> {| {{table}} | align="center" style="background:#f0f0f0;"|'''KAPA End repair mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''10x rxn mix''' |- | 10X End repai buffer||7.00||70.00 |- | KAPA End repair enzyme||3.00||30.00 |- | H2O||10.00||100.00 |- | Total||20.00||200.00 |} * Aliquot to column #1: 20ul (A & B: 40ul) - Add 20ul of End-repair reaction mix - Mix by pipetting 20 times - Shake at 1000 rpm for 5min (lid on) - Incubate at 20C for 30min - Add 120ul of diluted AMPure beads - Mix at 750rpm for 5min, 25C (lid on) - Incubate at 25C for 5 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads === A-tailing === * Total 30ul/reaction {| {{table}} | align="center" style="background:#f0f0f0;"|'''A-tailing reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''10x rxn mix''' |- | KAPA 10X A-tailing buffer||3.00||30.00 |- | KAPA A-tailing enzyme||2.00||20.00 |- | H2O||25.00||250.00 |- | Total||30.00||300.00 |} * Aliquot to column #2: 30ul (A & B: 60ul) - Add 30ul of A-tailing reaction mix - Mix by pipetting 20 times - Shake at 1000 rpm for 5min (lid on) - Incubate at 30C for 30min - Add 90ul of 20%PEG/2.5M NaCl - Mix at 750rpm for 5min, 25C (lid on) - Incubate at 25C for 5 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads === Adapter ligation === {| {{table}} | align="center" style="background:#f0f0f0;"|'''Total reaction in''' | align="center" style="background:#f0f0f0;"|'''50''' | align="center" style="background:#f0f0f0;"|'''ul''' |- | Ligation reaction mix||40||ul |- | Diluted Methylation adapters||10||ul |}<br> {| {{table}} | align="center" style="background:#f0f0f0;"|'''Ligation reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''10x rxn mix''' |- | KAPA 5X Ligation buffer||10.00||100.00 |- | KAPA DNA ligase||3.00||30.00 |- | H2O||27.00||270.00 |- | Total||40.00||400.00 |} * Aliquot ligation reaction mix to column #3: 40ul (A & B: 80ul) '''Methylation adapter preparation'''<br> 1. Use the methylated adapter with paired-end PCR primer pairs(PCR primer PE1.0 and PCR primer PE 2.0) provided in illumina's paired end genomic DNA sample prep kit 2. Use 10ul of methylated adapter to 5ug gDNA insert, if starting DNA amount is less than 5ug, titrate the volume of adapter reagent accordingly to maintain 10:1 ratio of DNA Since I used 0.2ug of DNA, used 0.2ul of adapters, but dilute to final volume 10ul (This volume is larger than KAPA protocol, but the total amount is the same) {| {{table}} | Prepare adapter||0.2ulx 10rxn||2.00 |- | H2O||9.8ulx10rxn||98.00 |- | Total||||100 |} * Aliquot diluted methylation adapter to column #4: 10ul (A & B: 20ul) - Add 40ul of Ligation reaction mix - Mix by pipetting 10 times - Add 10ul of diluted Methylation adapters - Mix by pipetting 10 times - Shake at 1000 rpm for 5min (lid on) - Incubate at 20C for 15min - Add 50ul of 20%PEG/2.5M NaCl - Mix at 750rpm for 5min, 25C (lid on) - Incubate at 25C for 5 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads - Resuspend adapter ligated DNA with ~23ul EB buffer == qPCR to check if the end repair, A-tailing, and adapter ligation work == {| {{table}} border = 1 | width="120pt" align="center" style="background:#f0f0f0;"|'''Components''' | width="50pt" align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''11.5 rxn mix''' |- | Adapter ligated DNA||1.00||0.00 |- | 10uM PCR_F||0.50||5.75 |- | 10uM PCR_R.Ind2||0.50||5.75 |- | 2x iQ MM||12.50||143.75 |- | H2O||10.50||120.75 |- | Total||25.00||276.00 |} * Aliquot 24ul, add 1ul of adapter ligated DNA '''Program''' 98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min --> 15C Hold [[File:20120826_testqPCR_adapterligated_N37.png| 600px]] - Load 2ul of PCR product to check amplification [[File:20120826_testqPCR_adapterligated_N37.jpg| 500px]] == Bisulfite conversion using EZ-96 DNA Methylation-Lightning™ MagPrep == * Performed bisulfite conversion manually * Elute with 40ul Elution buffer (final volume ~35ul) == Amplification of bisulfite sequencing libraries with N2 barcoded primers == * Perform in 50ul reaction with PCR_R.Ind1-10, add 7ul of adapter-ligated bis-cvt DNA {| {{table}} | Width="200pt" align="center" style="background:#f0f0f0;"|'''Components''' | width="50pt" align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''11.5 rxn mix''' |- | Adapter ligated bis-cvt DNA||7.00||0.00 |- | 10uM PCR_F||1.00||11.50 |- | 10uM PCR_R.Ind1-8, 21,22||1.00||0.00 |- | 2x iQ MM||25.00||287.50 |- | H2O||16.00||184.00 |- | Total||50.00||483.00 |} * Aliquot 42ul, add 1ul PCR_R.IndX and 7ul of adapter ligated bis-cvt DNA '''Program''' 98C 2min --> (98C 15sec -> 64C 20sec -> 72C 45sec) x 10 cycles --> 72C 3min --> 15C Hold '''qPCR result''' [[File:20120827_qPCR_WGBS_N37_50ul.png| 600px]] * Performed qPCR in total volume 100ul by using the same condition as above (in duplicated) {| {{table}} | Width="200pt" align="center" style="background:#f0f0f0;"|'''Components''' | width="50pt" align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''21.5 rxn mix''' |- | Adapter ligated bis-cvt DNA||14.00||0.00 |- | 10uM PCR_F||2.00||43.00 |- | 10uM PCR_R.Ind1-8,21,22||2.00||0.00 |- | 2x iQ MM||50.00||1075.00 |- | H2O||32.00||688.00 |- | Total||100.00||1806.00 |} * Aliquot 84ul, add 2ul PCR_R.IndX and 14ul of adapter ligated bis-cvt DNA '''Program''' 98C 2min --> (98C 15sec -> 64C 20sec -> 72C 45sec) x 10 cycles --> 72C 3min --> 15C Hold '''qPCR results''' [[File:20120827_qPCR_WGBS_N37_rep1-2.png| 600px]] {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Labeling''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | N37-Cerebellum (CRBL)||N37-1||PCR_R.N2Indx1 |- | N37-Colon||N37-2||PCR_R.N2Indx2 |- | N37-Frontal lobe (FL)||N37-3||PCR_R.N2Indx3 |- | N37-Heart||N37-4||PCR_R.N2Indx4 |- | N37-Small intestine (SI)||N37-5||PCR_R.N2Indx5 |- | N37-Liver||N37-6||PCR_R.N2Indx6 |- | N37-Lung||N37-7||PCR_R.N2Indx7 |- | N37-Skeletal muscle (SM)||N37-8||PCR_R.N2Indx8 |- | N37-Pancreas||N37-9||PCR_R.N2Indx21 |- | N37-Stomach||N37-10||PCR_R.N2Indx22 |} - Purify all PCR products (total volume 250ul) with 0.8 volume of 3x diluted AMPure bead by EPmotion - Elute with 70ul EB buffer - Perform PAGE quantification by loading 1ul of AMPure bead purified DNA [[File:ZhangLab_2 2012-08-27 17hr 22min_PQ_bead_WGBS_N37.jpg| 500px]] == PAGE quantification of 400-600bp fragments == [[File:ZhangLab_2 2012-08-27 17hr 40min_PQ_400-600bp.jpg| 500px]] {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 70ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 150ng''' |- | N37-1||4.48||313.30||33.51 |- | N37-2||2.81||196.92||53.32 |- | N37-3||4.33||302.97||34.66 |- | N37-4||4.41||308.72||34.01 |- | N37-5||4.04||282.85||37.12 |- | N37-6||5.03||351.84||29.84 |- | N37-7||4.74||332.11||31.62 |- | N37-8||3.99||279.46||37.57 |- | N37-9||4.11||287.69||36.50 |- | N37-10||4.54||317.72||33.05 |} - Combine 150ng of each library - Perform PAGE size-selection of the size range 400-600bp in 6% TBE 5-well gel (2 gels) [[File:ZhangLab_2 2012-08-27 19hr 11min_PAGE-SS_WGBS_N37.png| 500px]] - Resuspend with H2O, total volume 45ul == dsDNA Qubit HS quantification == {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Library IDs''' | align="center" style="background:#f0f0f0;"|'''Concentration 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;"|'''Final conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Ave. Conc. (ng/ul)''' |- | AL-exome_Aug21 1ul||46.60||ng/mL||1.00||200||9.32||9.76 |- | AL-exome_Aug21 2ul||102.00||ng/mL||2.00||100||10.20|| |- | NP_WGBS_N37_Aug26 1ul||18.70||ng/mL||1.00||200||3.74||3.80 |- | NP_WGBS_N37_Aug26 2ul||38.50||ng/mL||2.00||100||3.85|| |- | NP-BSPP-GL-AL_Ind9-29_Aug23 1ul||72.50||ng/mL||1.00||200||14.50||14.95 |- | NP-BSPP-GL-AL_Ind9-29_Aug23 2ul||154.00||ng/mL||2.00||100||15.40|| |} <br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Library IDs''' | align="center" style="background:#f0f0f0;"|'''Ave. Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Size (bp)''' | align="center" style="background:#f0f0f0;"|'''Conc. (nM)''' | align="center" style="background:#f0f0f0;"|'''MW (g/mole)''' |- | AL-exome_Aug21||9.76||300||53.52||182,377.9 |- | NP_WGBS_N37_Aug26||3.80||500||12.49||303,857.9 |- | NP-BSPP-GL-AL_Ind9-29_Aug23||14.95||375||65.59||227,932.9 |} '''Jeff' libraries'''<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Library IDs''' | align="center" style="background:#f0f0f0;"|'''Concentration 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;"|'''Final conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Size (bp)''' | align="center" style="background:#f0f0f0;"|'''Conc. (nM)''' | align="center" style="background:#f0f0f0;"|'''MW (g/mole)''' |- | JG-pool1||3.44||ng/mL||5||40||0.14||450||0.50||273,487.9 |- | JG-pool2||2.56||ng/mL||5||40||0.10||450||0.37||273,487.9 |} - Quantified concentration by Qubit dsDNA assay HS (based on size 375bp): 3.80ng/ul pr 12.49nM [[File:ZhangLab_2 2012-08-28 14hr 52min_PQ_NP_WGBS_N37.png| 200px]] - Concentration by PAGE quantification is 14.8ng/ul * '''Library IDs: NP_WGBS_N37_Aug26, 2012, sequenced on HiSeqAug28 run at Biogem (Lane : 4-6)'''
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