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= Preparation of RRBS sequencing libraries of solid tumor tissue sample from colon and lung cancer patients = [[noi:DMR220k_LabNotes#2014| '''Calendar''']] ==== Sample info ==== * [[noi:Samples_from_Moores_Cancer_Center#2014-06-27| Solid tumor tissue from colon cancer patients (006-T)]] * [[noi:Samples_from_Moores_Cancer_Center#2014-06-27| Solid tumor tissue from lung cancer patients (007-T)]] {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Project ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Zhang lab ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' |- | UCSD-006-1||6-T-1||43.40 |- | UCSD-006-2||6-T-2||31.60 |- | UCSD-006-3||6-T-3||31.50 |- | UCSD-006-4||6-T-4||41.70 |- | UCSD-006-5||6-T-5||30.50 |- | UCSD-007-1||7-T-1||30.70 |- | UCSD-007-2||7-T-2||44.60 |- | UCSD-007-3||7-T-3||35.90 |- | UCSD-007-4||7-T-4||33.80 |- | UCSD-007-5||7-T-5||20.40 |} * X-Y_Z: ** X = project ID number. 4 = pancreas, 6 = colon, 7 = lung ** Y = sample type. T = tissue, P = plasma, S = serum ** Z = sample ID == 1) MspI digestion == * Conditions: Digest 100ng of DNA with 20U of MspI in 30ul reaction at 37C O/N (normally 3h) and heat inactivate at 65C 20min * For 100ng DNA input, I used 2ul (20U) of MspI. * I spike in 1% of unmeth lambda DNA (1ng) * I actually start MspI at late night of 2014-06-27, so I incubated it O/N as it doesn't hurt to incubate DNA with MspI longer than 3h. * I do two replicates for each sample <br> ==== <u>MspI digestion set up</u> ==== 1.1) Colon samples, UCSD-006<br> {| {{table}} border = 1 | align="center" style="width:40px;background:#f0f0f0;"|'''#''' | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:70px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:70px;background:#f0f0f0;"|'''Volume for 100ng (ul)''' | align="center" style="width:70px;background:#f0f0f0;"|'''H2O to 10ul''' | align="center" style="width:70px;background:#f0f0f0;"|'''MspI Mix''' | align="center" style="width:70px;background:#f0f0f0;"|'''Total''' | align="center" style="width:80px;background:#f0f0f0;"|'''Code''' |- | 1||6-T-1.1||43.40||2.30||7.70||20.00||30.00||6T-1.1 |- | 2||6-T-1.2||43.40||2.30||7.70||20.00||30.00||6T-1.2 |- | 3||6-T-2.1||31.60||3.16||6.84||20.00||30.00||6T-2.1 |- | 4||6-T-2.2||31.60||3.16||6.84||20.00||30.00||6T-2.2 |- | 5||6-T-3.1||31.50||3.17||6.83||20.00||30.00||6T-3.1 |- | 6||6-T-3.2||31.50||3.17||6.83||20.00||30.00||6T-3.2 |- | 7||6-T-4.1||41.70||2.40||7.60||20.00||30.00||6T-4.1 |- | 8||6-T-4.2||41.70||2.40||7.60||20.00||30.00||6T-4.2 |- | 9||6-T-5.1||30.50||3.28||6.72||20.00||30.00||6T-5.1 |- | 10||6-T-5.2||30.50||3.28||6.72||20.00||30.00||6T-5.2 |- | 11||6-T-NTC||0.00||0.00||10.00||20.00||30.00||6T-NTC |} 1.2) Lung sample, UCSD-007<br> {| {{table}} border = 1 | align="center" style="width:40px;background:#f0f0f0;"|'''#''' | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:70px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:70px;background:#f0f0f0;"|'''Volume for 100ng (ul)''' | align="center" style="width:70px;background:#f0f0f0;"|'''H2O to 10ul''' | align="center" style="width:70px;background:#f0f0f0;"|'''MspI Mix''' | align="center" style="width:70px;background:#f0f0f0;"|'''Total''' | align="center" style="width:80px;background:#f0f0f0;"|'''Code''' |- | 1||7-T-1.1||30.70||3.26||6.74||20.00||30.00||7T-1.1 |- | 2||7-T-1.2||30.70||3.26||6.74||20.00||30.00||7T-1.2 |- | 3||7-T-2.1||44.60||2.24||7.76||20.00||30.00||7T-2.1 |- | 4||7-T-2.2||44.60||2.24||7.76||20.00||30.00||7T-2.2 |- | 5||7-T-3.1||35.90||2.79||7.21||20.00||30.00||7T-3.1 |- | 6||7-T-3.2||35.90||2.79||7.21||20.00||30.00||7T-3.2 |- | 7||7-T-4.1||33.80||2.96||7.04||20.00||30.00||7T-4.1 |- | 8||7-T-4.2||33.80||2.96||7.04||20.00||30.00||7T-4.2 |- | 9||7-T-5.1||20.40||4.90||5.10||20.00||30.00||7T-5.1 |- | 10||7-T-5.2||20.40||4.90||5.10||20.00||30.00||7T-5.2 |- | 11||7-T-NTC||0.00||0.00||10.00||20.00||30.00||7T-NTC |} ==== <u>Msp I Reaction Mix</u> ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''24 rxn Mix''' |- | MspI (10U/ul)||2.00||48.00 |- | 10x Tango Buffer||3.00||72.00 |- | 1ng/ul unmeth-lambda DNA||1.00||24.00 |- | H2O||14.00||336.00 |- | Total||20.00|| |} '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Aliquot MspI reaction mix to 8-tube strip. 65ul for tube #1-3, #6-8 and 44ul for tube #4-5 :- Add H2O to each tube :- Add 100ng of DNA to each tube :- Add 20ul of MspI reaction Mix to each tube and pipette few times :- Spin down the tubes :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tubes :- 37C, O/N (~13.5h) --> 65C, 20min --> hold at 4C (set heat lid)<br> <br> == 2) End-repair/dA-tailing == '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Mix 26ul of Klenow exo- with 26ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA<br> - Aliquot Klenow, exo- and dA:dC:dG mix to 8-tube strip. 7ul for tube #1-3, #6-8 and 5ul for tube #4-6 :- Add 2ul of Klenow, exo- and dA:dC:dG mix to each tube of MspI digested DNA with multichannel pipette. Pipette few times :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid) :- 75C, 10min --> hold at 4C (heat lid on) * Before set up ligation reaction, purify end-repaired DNA with 2x volume AMPure beads (64ul each) and resuspend the bead with 20ul EB buffer ==== <u>AMPure bead purification</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Freshly prepare 8mL of 75% EtOH by mixing 6mL of 100% EtOH with 2mL of H2O (prepare when the tubes sit on magnet)<br> - Aliquot 1400ul of AMPure beads in 30mL reservoir and leave at RT<br> :- Add 64ul AMPure bead with multichannel pipette. Mix by pipetting 10x :- Wait for 30min :- Transfer to sit on magnet for 5min :- Wash twice with 180ul freshly prepared 75% EtOH ::- After adding 75% EtOH (1st time), wait for 30sec before pipetting up & down 5x :- Remove all 75% EtOH :- Dry the bead for 20min :- Resuspend with 20 EB Buffer <u>''(keep the beads in the tubes!)''</u><br> <br> == 3) Methylated adaptor ligation == * Conditions: Add 1ul of non-diluted TruSeq adaptors (do not know actual concentration of adaptors) in 30ul ligation reaction with 6000U of T4 DNA ligase ==== <u>Ligation reaction set up</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Thaw TruSeq adaptor on ice (during drying the beads, keep it on ice all the time)<br> - Prepare ligation reaction mix (during drying the beads) ====<u> Ligation reaction Mix</u> ==== :{| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:70px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:70px;background:#f0f0f0;"|'''24rxn Mix''' |- | dA-tailed DNA||20.00||0.00 |- | 10x Ligation Buffer||3.00||72.00 |- | T4 DNA Ligase (30U/ul)||1.00||24.00 |- | TruSeq Adaptor DNA||1.00||24.00 |- | H2O||5.00||120.00 |- | Total||30.00|| |} '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Aliquot ligation reaction mix to 8-tube strip. 29ul for tube #1-3, #6-8 and 20ul for tube #4-5 :- Add 1ul of non-diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction :{| {{table}} class = wikitable | align="center" style="width:90px;background:dodgerblue;"|'''Sample''' | align="center" style="width:70px;background:magenta;"|'''TruSeq Index''' | align="center" style="width:90px;background:dodgerblue;;"|'''Sample''' | align="center" style="width:70px;background:magenta;"|'''TruSeq Index''' |- | 6-T-1.1||1||7-T-1.1||13 |- | 6-T-1.2||2||7-T-1.2||14 |- | 6-T-2.1||23||7-T-2.1||15 |- | 6-T-2.2||4||7-T-2.2||16 |- | 6-T-3.1||5||7-T-3.1||27 |- | 6-T-3.2||6||7-T-3.2||18 |- | 6-T-4.1||7||7-T-4.1||19 |- | 6-T-4.2||8||7-T-4.2||20 |- | 6-T-5.1||25||7-T-5.1||21 |- | 6-T-5.2||10||7-T-5.2||22 |- | 6-T-NTC||10||7-T-NTC||22 |} :- Add 9ul of ligation reaction mix with multichannel pipette :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 16C, 24h (no heat lid. Ligation time is flexible. I usually do at least 20h at 16C) :- Mix by gentle pulse-vortexing on PCR rack 5x :- Quickly spin down :- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on) * Before continue to bisulfite conversion, purify adaptor-ligated DNA with 2x volume diluted AMPure beads (60ul each) and resuspend the bead with 31ul EB buffer<br> ==== 2014-06-29 ==== ==== <u>AMPure bead purification</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Freshly prepare 8mL of 75% EtOH by mixing 6mL of 100% EtOH with 2mL of H2O (prepare when the tubes sit on magnet)<br> - Aliquot 670ul of AMPure beads and 670ul of 20% PEG 8000/5M NaCl in 1.5mL tube, mix well and leave at RT (forgot to dilute, used beads directly)<br> :- Add 60ul AMPure bead with multichannel pipette. Mix by pipetting 10x :- Wait for 30min :- Transfer to sit on magnet for 5min :- Wash twice with 180ul freshly prepared 75% EtOH ::- After adding 75% EtOH (1st time), wait for 30sec before pipetting up & down 5x :- Remove all 75% EtOH :- Dry the bead for 20min :- Resuspend with 31 EB Buffer. :- Wait for 2min. :- Transfer to sit on magnet for 2 min and transfer 30ul of adaptor-ligated DNA to new strip tubes for bisulfite conversion <u>(discard the beads)</u> :- Continue to bisulfite conversion <br> == Bisulfite conversion == * Performed bisulfite conversion using LifeTech MethylCode™ Bisulfite Conversion Kit and did the same procedure following manufacturer's instruction and elute with 30ul Elution buffer. '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> :- Prepare 2 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer :- Mix well and spin down the tube :- Put on thermomixer at 37C for 15min, 1,000rpm :- Solution should be clear :- Spin down the tube :- Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA --> total volume 150ul :- Mix by pipetting 10X with multi-channel pipette :- Spin down the tube :- Incubate following below program ::- 98°C for 10 minutes (DNA denaturation) ::- 64°C for 2.5 hours (Bisulfite conversion) ::- 4°C storage for up to 20 hours or continue to desulfonation :- Add 600ul Binding Buffer to each column :- Adjust p200 to 200ul and transfer bisulfite treated DNA to the column :- Mix by pipetting 5x :- Spin down column at 14,000 rpm for 30s :- <u>Discard spnt</u> :- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube) :- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u> :- Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube) :- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u> :- Add 200ul Wash Buffer :- Spin down column at 14,000 rpm for 30s :- <u>Discard spnt</u> :- Add 200ul Wash Buffer :- Spin down column at 14,000 rpm for 3min <u>(do not discard spnt)</u> :- Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transferring to new tube :- Add 38ul of Elution Buffer (warm Elution Buffer at 60C for 10min) :- Wait for 2min :- Spin down column at 12,000 rpm for 1min :- Continue to amplification (During elution step, PCR mix should be prepared) == Amplification == === TEST PCR === {| {{table}} border = 1 | align="center" style="width:160px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc./amount''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="bwidth:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''25 rxn mix''' |- | Bis-cvt DNA||||||||||6.00||0.00 |- | 10X Reaction buffer||10||X||1||X||2.00||50.00 |- | dNTP mix||10||mM||0.25||mM||0.50||12.50 |- | TruS_F/R||10||uM||0.3||uM||0.60||15.00 |- | 50X SYBG||50||X||0.8||X||0.32||8.00 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||0.40||10.00 |- | H2O||||||||||10.18||254.50 |- | Total ||||||||||20.00|| |} :- Aliquot 14 + 6ul DNA template :- Mix well. Split 2X of 8.5ul :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X [14X & 16X] --> 72C for 2min'' * There was error to realplex during annealing step of 15 cycles. I needed to remove samples. I loaded only 14X to save the gel and plan to loaded 16 cycles PCR if the intensity of 14X is too low. :- Add 10ul of 2X loading dye to PCR product and load 10ul PCR product/dyemix in 12% gel and run at 250 volts for 40min '''Colon tumor samples''' [[File:ZhangLab_2 2014-06-30 17hr 16min_6T_100ngRRBS_TEST-14X.jpg| 520px]] '''Lung tumor samples''' [[File:ZhangLab_2 2014-06-30 17hr 16min_7T_100ngRRBS_TEST-14X-e.jpgg| 520px]] * I do not know why sample 7-T_3.2 and 7-T_4.1 failed. May need to check with Dr. Zhang if he want to sequence the two duplicates or only 1 library for each sample. * No need to load 16 cycles PCR since 14 cycles look good and not too faint or over-amplified. * I need to repeat 7-T_3.2 and 7-T_4.1. I plan to repeat both of them in two duplicates. * Continued to prepare sequencing library by doing PCR in 100ul reaction with 30% BIS-DNA. {| {{table}} border = 1 | align="center" style="width:160px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc./amount''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''20.5''' |- | Bis-cvt DNA||||||||||30.00||0.00 |- | 10X Reaction buffer||10||X||1||X||10.00||205.00 |- | dNTP mix||10||mM||0.25||mM||2.50||51.25 |- | TruS_F/R||10||uM||0.3||uM||3.00||61.50 |- | 50X SYBG||50||X||0.8||X||1.60||32.80 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||2.00||41.00 |- | H2O||||||||||50.90||1043.45 |- | Total ||||||||||100.00|| |} :- Aliquot 70 + 30ul DNA template :- Mix well. Split 2X of 50ul (actual ~48ul) :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 14 cycles --> 72C for 2min'' :- Stopped at 14X cycles based on TEST PCR :- I pooled the two PCR replicates (total ~90ul for each samples) and purified with 1x volume AMPure beads and eluted with 55ul EB buffer :- I did PAGE quantification by loading 3ul of bead-purified sequencing libraries [[File:ZhangLab_2 2014-07-02 19hr 34min_PQ_6T_100ngRRBS.jpg| 500px]] [[File:ZhangLab_2 2014-07-02 19hr 34min_PQ_7T_100ngRRBS.jpg|500px]] * It's so wired for sample 6T_4.2 since it looked fine in TEST PCR, but it's very faint after amplification in 50ul and bead purified. '''Repeat smaple 7T_4.1 (also include 7T_4.2)''' [[File:ZhangLab_2 2014-07-03 17hr 35min_PQ_repeat_7T_4.jpg| 300px]] * I reloaded sample 6T_2.1 and 6T_4.2 again. To confirm that they had very low concentration. ==== PAGE quantification results ==== <u> 006-T samples</u> {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)''' |- | 6-T-1.1||1||6.53||326.73 |- | 6-T-1.2||2||9.91||495.56 |- | 6-T-2.1||23||2.00||100.20 |- | 6-T-2.2||4||7.14||356.87 |- | 6-T-3.1||5||10.60||530.01 |- | 6-T-3.2||6||6.70||334.87 |- | 6-T-4.1||7||6.19||309.49 |- | 6-T-4.2||8||0.71||35.55 |- | 6-T-5.1||25||4.31||215.60 |- | 6-T-5.2||10||8.26||412.83 |} <u> 007-T samples</u> * Value of sample 7-T-4.1 is the repeated sample (will be added) {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)''' |- | 7-T-1.1||13||5.12||255.78 |- | 7-T-1.2||14||3.65||182.39 |- | 7-T-2.1||15||4.18||208.94 |- | 7-T-2.2||16||6.55||327.39 |- | 7-T-3.1||27||2.70||135.19 |- | 7-T-3.2||18||3.72||185.97 |- | <span style="color:crimson">7-T-4.1</span>||19||9.30||465.00 |- | 7-T-4.2||20||3.59||179.72 |- | 7-T-5.1||21||5.76||288.10 |- | 7-T-5.2||22||3.82||190.84 |}<br> ==== <u>Library pooling</u> ==== =====<span style="color:mediumblue"><u> 006-T samples</u></span>===== * Since I may need to sequence only one replicate, I combine sequencing libraries into two set; set 1 and set2. Set 1 set the first priority, so I included sample 6-T-2_2 in set one as sample 6-T-2_1had very faint signal. I included sample 6-T_2.1 in set 2 <span style="color:mediumblue">NP-RRBS-6T-set1_100ng-Jun28</span> {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:60px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 120ng (ul)''' |- | 6-T_1.1||1||6.53||18.36 |- | <span style="color:crimson">6-T_2.2</span>||4||7.14||16.81 |- | 6-T-3.1||5||10.60||11.32 |- | 6-T-4.1||7||6.19||19.39 |- | 6-T-5.1||25||4.31||27.83 |} :- Add 20ul of 6X loading dye and load 37.9ul in 3 well of 5-well 6% TBE gel <span style="color:mediumblue">NP-RRBS-6T-set2_100ng-Jun28</span> * Set 2 I include 4 libraries and do PAGE size selection of sample 6-T-4.2 separately since it had very faint smear. {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:60px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)''' |- | 6-T-1.2||2||9.91||10.09 |- | 6-T-2.1||23||2.00||49.90 |- | 6-T-3.2||6||6.70||14.93 |- | 6-T-5.2||10||8.26||12.11 |} :- Add 20ul of 6X loading dye and load 35.68 in 3 well of 5-well 6% TBE gel <span style="color:mediumblue">NP-RRBS-6T-4_2-set2_100ng-Jun28</span> {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:60px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' |- | 6-T-4.2||8||0.71||50.00 |} :- Add 10ul of 6X loading dye and load 60ul in 1 well of 5-well 6% TBE gel =====<span style="color:red"><u> 007-T samples</u></span>===== <span style="color:red">NP-RRBS-7T-set1_100ng-Jun28</span> {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:60px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)''' |- | 7-T-1.1||13||5.12||19.55 |- | 7-T-2.1||15||4.18||23.93 |- | 7-T-3.1||27||2.70||36.98 |- | 7-T-4.1||19||9.30||10.75 |- | 7-T-5.1||21||5.76||17.35 |} :- Add 25ul of 6X loading dye and load 44.52ul in 3 well of 5-well 6% TBE gel <span style="color:red">NP-RRBS-7T-set2_100ng-Jun28</span> {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:60px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)''' |- | 7-T-1.2||14||3.65||27.41 |- | 7-T-2.2||16||6.55||15.27 |- | 7-T-3.2||18||3.72||26.89 |- | 7-T-4.2||20||3.59||27.82 |- | 7-T-5.2||22||3.82||26.20 |} :- Add 30ul of 6X loading dye and load 51.2ul in 3 well of 5-well 6% TBE gel<br> ==== PAGE size selection ==== [[File:ZhangLab_2 2014-07-06 17hr 56min_gel1.jpg|380px]] - NP-RRBS-6T_set1_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification [[File:ZhangLab_2 2014-07-06 17hr 58min_gel2.jpg|380px]] - NP-RRBS-6T_set2_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification - NP-RRBS-6T_4_2_set2_100ng-Jun28: Resuspend with 12ul H2O x 1tube--> volume 12ul, no PAGE verification [[File:ZhangLab_2 2014-07-06 18hr 03min_gel3.jpg|380px]] - NP-RRBS-7T_set1_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification [[File:ZhangLab_2 2014-07-06 18hr 04min_gel4.jpg|380px]] - NP-RRBS-7T_set2_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification ==== PAGE verification ==== ---- === 2014-07-08 === ==== Preparing sequencing library pool for IGM ==== * We will do HiSeq Rapid run, PE 100+7bp * Spike in with 5% PhiX in each lane :1). '''NP_RRBS_6T_100ng_20140708''' (Loaded in lane 1) :{| {{table}} class = wikitable | align="center" style="width:220px;background:#f0f0f0;"|'''Seq. Library''' | align="center" style="width:60px;background:#f0f0f0;"|'''# of ind. Library''' | align="center" style="width:80px;background:#f0f0f0;"|'''# of read for each (M)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total # of reads (M)''' | align="center" style="width:60px;background:#f0f0f0;"|'''Loading %''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (nM)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Ratio of % to 6T-set1''' | align="center" style="width:80px;background:#f0f0f0;"|'''# of mole to pool (nmole)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume to pool (ul)''' |- | NP-RRBS-6T_set1_100ng-Jun28||5||10||50||50||11.63||1||50||4.30 |- | NP-RRBS-6T_set2_100ng-Jun28||4||10||40||40||9.01||0.8||40||4.44 |- | NP-RRBS-6T_4_2_set2_100ng-Jun28||1||10||10||10||2.44||0.2||10||4.10 |- | ||||||||||||Total||100||12.84 |- | ||||||||||||Final conc.||7.79||nM |} :2). '''NP_RRBS_7T_100ng_20140708''' (Loaded in lane 2) :{| {{table}} class = wikitable | align="center" style="width:220px;background:#f0f0f0;"|'''Seq. Library''' | align="center" style="width:60px;background:#f0f0f0;"|'''# of ind. Library''' | align="center" style="width:80px;background:#f0f0f0;"|'''# of read for each (M)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total # of reads (M)''' | align="center" style="width:60px;background:#f0f0f0;"|'''Loading %''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (nM)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Ratio of % to 7T-set1''' | align="center" style="width:80px;background:#f0f0f0;"|'''# of mole to pool (nmole)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume to pool (ul)''' |- | NP-RRBS-7T_set1_100ng-Jun28||5||10||50||50||15.6||1||100||6.41 |- | NP-RRBS-7T_set2_100ng-Jun28||5||10||50||50||13.47||1||100||7.42 |- | ||||||||||||Total||200||13.83 |- | ||||||||||||Final conc.||14.46||nM |}
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