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= RRBS library prep, mouse hippocampus gDNA (Zhou's lab) = [[noi:DMR220k_LabNotes#2014|'''Back to calendar''']]<br> * 2014-12-01: Received 9 mouse hippocamplus gDNA from Zhou's lab === N.D. measurement by Orit === {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''ng/ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''A260''' | align="center" style="width:80px;background:#f0f0f0;"|'''A280''' | align="center" style="width:80px;background:#f0f0f0;"|'''260/280''' | align="center" style="width:80px;background:#f0f0f0;"|'''260/230''' |- | C1||113.72||2.274||1.199||1.9||2.32 |- | C2||84.47||1.689||0.919||1.84||2.19 |- | C3||109.47||2.189||1.207||1.81||1.79 |- | C4||116.84||2.337||1.273||1.84||2.23 |- | H1||115.85||2.317||1.285||1.8||2.14 |- | H2||107.32||2.146||1.161||1.85||2.11 |- | H3||106.12||2.122||1.14||1.86||2.11 |- | H3||90.76||1.815||0.997||1.82||2.17 |- | H4||145.22||2.904||1.68||1.73||1.29 |- | H5||96.04||1.921||1.042||1.84||2.2 |} * I re-quantified concentration by Qubit dsDNA HS assay using 1ul for quantification === Qubit dsDNA HS quantification result === {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:60px;background:#f0f0f0;"|'''uL used''' | align="center" style="width:80px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' |- | HC-C1||459||ng/mL||1||200||91.80 |- | HC-C2||382||ng/mL||1||200||76.40 |- | HC-C3||510||ng/mL||1||200||100.00 |- | HC-C4||510||ng/mL||1||200||100.00 |- | HC-H1||487||ng/mL||1||200||97.40 |- | HC-H2||495||ng/mL||1||200||99.10 |- | HC-H3||395||ng/mL||1||200||79.00 |- | HC-H4||493||ng/mL||1||200||98.60 |- | HC-H5||428||ng/mL||1||200||85.60 |} == 1) MspI digestion == * I will use 500ng for RRBS library prep and spike in with 1ng of unmethylated lambda DNA for monitoring bisulfite conversion rate * Conditions: Digest 500ng of DNA with 20U of MspI in 30ul reaction at 37C 3h and heat inactivate at 65C 20min ==== <u>MspI digestion set up</u> ==== {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 500ng (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample code''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O''' | align="center" style="width:80px;background:#f0f0f0;"|'''MspI Mix''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total (ul)''' |- | HC-C1||91.80||5.45||C1||4.55||20.00||30.00 |- | HC-C2||76.40||6.54||C2||3.46||20.00||30.00 |- | HC-C3||100.00||5.00||C3||5.00||20.00||30.00 |- | HC-C4||100.00||5.00||C4||5.00||20.00||30.00 |- | HC-H1||97.40||5.13||H1||4.87||20.00||30.00 |- | HC-H2||99.10||5.05||H2||4.95||20.00||30.00 |- | HC-H3||79.00||6.33||H3||3.67||20.00||30.00 |- | HC-H4||98.60||5.07||H4||4.93||20.00||30.00 |- | HC-H5||85.60||5.84||H5||4.16||20.00||30.00 |- | NTC||0.00||0.00||NTC||10.00||20.00||30.00 |} ==== <u>Msp I reaction Mix</u> ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1 rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''10.5 rxn mix''' |- | MspI (10U/ul)||2.00||21.00 |- | 10X Tango Buffer||3.00||31.50 |- | 1ng/ul unmeth-lambda DNA||1.00||10.50 |- | H2O||14.00||147.00 |- | Total||20.00|| |} :- Add H2O to each tube :- Add 500ng 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, 3h --> 65C, 20min --> hold at 4C (set heat lid)<br> <br> == 2) End-repair/dA-tailing == * After MspI, I took 2ul of MspI-digested DNA for gel verification (equal to DNA ~33.33ng). Total volume left is 28ul * Mix 13ul of 5U/ul Klenow exo- (Thermoscientific) with 13ul of 20:2:2mM dAdCdG * Add 2ul of Klenow exo-/dAdCdG mix to each tube. Total volume is 30ul :- Spin down the tubes :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tubes :- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid) :- 75C, 10min --> hold at 4C (heat lid on) :- Perform AMPure bead purification with 2x volume AMPure beads ==== <u>AMPure bead purification</u> ==== :- Add 60ul AMPure bead. Mix by pipetting 10x :- Wait for 15min :- Transfer to sit on magnet for 5min :- Wash twice with 200ul freshly prepared 80% EtOH ::- After adding 80% EtOH (1st time), wait for 30sec before pipetting up & down 5x :- Remove all 80% EtOH :- Dry the bead for 15min :- Resuspend with 20 EB Buffer <u>''(keep the beads in the tubes!)''</u> == Methylated adaptor ligation == {| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''11rxn Mix''' |- | dA-tailed DNA||20.00||0.00 |- | 10x Ligation Buffer||3.00||33.00 |- | T4 DNA Ligase (6000U/ul)||1.00||11.00 |- | TruSeq Adaptor (15uM)||1.50||0.00 |- | H2O||4.50||49.50 |- | Total||30.00|| |} :- Add 1.5ul of 15uM (non-diluted adaptor) TruSeq adaptor to each tube :{| {{table}} class = wikitable | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Truseq Index''' |- | HC-C1||1 |- | HC-C2||2 |- | HC-C3||3 |- | HC-C4||4 |- | HC-H1||5 |- | HC-H2||6 |- | HC-H3||7 |- | HC-H4||8 |- | HC-H5||9 |- | NTC||10 |} * Final conc. of adaptor in 30ul lgation reaction is 750nM :- Add 8.5ul of ligation reaction mix with adaptors :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 16C, 24h (no heat lid) :- 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 AMPure beads (60ul each) and resuspend the bead with 21ul EB buffer ==== <u>AMPure bead purification</u> ==== :- Add 60ul AMPure bead. Mix by pipetting 10x :- Wait for 15min :- Transfer to sit on magnet for 5min :- Wash twice with 200ul freshly prepared 80% EtOH ::- After adding 80% EtOH (1st time), wait for 30sec before pipetting up & down 5x :- Remove all 80% EtOH :- Dry the bead for 15min :- Resuspend with 21 EB Buffer :- Wait for 2min. :- Transfer to sit on magnet for 2 min and transfer 20ul of adaptor-ligated DNA to new strip tubes <u>(discard the beads)</u> :- Continue to bisulfite conversion === Bisulfite conversion === * Performed bisulfite conversion using the same procedure following manufacturer's instruction [http://www.zymoresearch.com/downloads/dl/file/id/490/d5030i.pdf| '''EZ DNA Methylation-Lightning™ Kit'''] and elute with 34ul Elution buffer. :- Add 130ul complete Lightning 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 (Magprep, Heated Lid) ::- 98°C for 10 minutes (DNA denaturation) ::- 54°C for 1 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) :- Wait for 18min :- 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 transfering to new tube :- Add 36ul 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 == Amplification == === TEST PCR === :- Prepare PCR in total 20ul reaction with 4 ul BIS-DNA {| {{table}} border = 1 | align="center" style="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.''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''10.5 rxn mix''' |- | Bis-cvt DNA||||||||||3.00||0.00 |- | 2X KAPA HiFi Uracil HotStart ReadyMix||2||X||1||X||5.00||52.50 |- | TruS_F/R||10||uM||0.3||uM||0.30||3.15 |- | 50X SYBG||50||X||0.4||X||0.08||0.84 |- | H2O||||||||||1.62||17.01 |- | Total||||||||||10.00|| |} :- Aliquot 3.5ul + 1.5ul BIS-DNA ::- ''98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 13X --> 72C for 1min'' :- Verify all PCR product in 6% TBE gel [[File:ZhangLab_2 2014-12-12 20hr 24min_RRBS-DZ-5ul.jpg|500px]] * It looked like the pattern of size distribution was different from previous experiment as the size seemed to be more larger than sequencing library prepared from human tumor DNA. * I continued to amplified the rest of BIS-DNA in 100ul reaction with cycle number 14 cycles for sample C1, C2, H1, H3, 15 cycles for samples C4, H4, H5 and 16 cycles for sample C3 :- Purified with 1X AMPure bead, elute with 32ul EB buffer :- PAGE quantify by loading 4ul [[File:ZhangLab_2 2014-12-15 19hr 52min_PQ_SeqLib-4ul.jpg| 500px]] {| {{table}} border = 1 | align="center" style="width:100px;background:#f0f0f0;"|'''Sample IDs''' | align="center" style="width:100px;background:#f0f0f0;"|'''TruSeq Index''' | align="center" style="width:100px;background:#f0f0f0;"|'''Seq (RC)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Con. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Yield in 28ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 75ng (ul)''' |- | HC-C1||1.00||ATCACG||6.66||186.53||11.26 |- | HC-C2||2.00||CGATGT||7.07||198.07||10.60 |- | HC-C3||3.00||TTAGGC||3.16||88.38||23.76 |- | HC-C4||4.00||TGACCA||7.95||222.62||9.43 |- | HC-H1||5.00||ACAGTG||8.53||238.81||8.79 |- | HC-H2||6.00||GCCAAT||7.86||220.06||9.54 |- | HC-H3||7.00||CAGATC||9.33||261.24||8.04 |- | HC-H4||8.00||ACTTGA||7.78||217.90||9.64 |- | HC-H5||9.00||GATCAG||6.64||186.03||11.29 |}
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