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= Integration of DNA SMART ChIP-Seq protocol with RRBS for low input DNA methylation assay = [[noi:DMR220k_LabNotes|'''Link to calendar''']] * Dr. Zhang suggested to try template switching protocol in stead of the standard adaptor ligation base method to generate bisulfite sequencing library. The idea is to fragment DNA by MspI to enrich only the targets for RRBS then perform bisulfite conversion on MspI-digested DNA. The single strand bisulfite converted DNA will be used as the template for the subsequent steps, including dephosphorylation, poly dT-tailng, first strand cDNA synthesis, and template switching to append the partial adaptor sequence to the newly synthesized cDNA. Then PCR with adaptor primers will be performed to amplified the libraries. One advantage of performing bisulfite conversion before generating sequencing library is that the template with ligated adaptors won't be damaged within the molecules as occurs in standard WGBS protocol. [[Media:DNA_SMART_ChIP-Seq_User_Manual_082014.pdf| '''DNA SMART ChIP-Seq kit Manual''']] == <span style="color:dodgerblue"><u>TEST #1</u></span> == * I used gDNA sample of cancer cell line CANC1 (eq. to XXXX) for the first TEST experiment before moving to gDNA from solid tumor tissue. * Vary amount of template at 1ng, 100pg and 10pg. :- Dilute 86.4ng/ul to 2ng/ul --> re-quantify conc. by Qubit: 1.57ng/ul :- Mix 2.32ul of 86.4ng/ul of CANC1 and 97.68ul of TE buffer :- Dilute to : 100pg/ul, 10pg/ul, 1pg/ul to used 10ul of each concentration for MspI digestion === <u>MspI reaction Mix</u> === ::{| {{table}} | style="width:180px;"|'''Components''' | style="width:100px;"|'''1x rxn''' | style="width:100px;"|'''4.5x rxn''' |- | MspI (10U/ul)||1.00||4.50 |- | 10x Tango Buffer||2.00||9.00 |- | H2O||7.00||31.50 |- | Total||10.00|| |} === <u>MspI digestion set up</u> === ::{| {{table}} border = 1 | style="width:120px;"|'''Input amount''' | style="width:80px;"|'''Stock used (pg/ul)''' | style="width:80px;"|'''Volume of stock (ul)''' | style="width:80px;"|'''MspI reaction mix''' | style="width:80px;"|'''Total (ul)''' |- | 1ng||100.00||10.00||10.00||20.00 |- | 100pg||10.00||10.00||10.00||20.00 |- | 10pg||1.00||10.00||10.00||20.00 |- | NTC||H2O||10.00||10.00||20.00 |} :- Add 10ul of DNA to each tube :- Add 10ul 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> === Bisulfite conversion === :- Perform bisulfite conversion by EZ-Lightning DNA Methylation kit using instructions provided by manufacturer without using tRNA as carrier. :- Elute with 12ul of Elution Buffer. The left over volume of DNA is ~10ul. :- Continue to the the steps of DNA SMART ChIP-Seq protocol <ve> '''<u> Note from DNA SMART ChIP-Seq kit</u>'''<br> '''Sample Requirements'''<br> This kit can generate sequencing libraries from 100 pg–10 ng of ssDNA or dsDNA up to 2 kb in length, but it has been optimized specifically for ChIP DNA with an average size of 200–400 bp. If your starting material is significantly larger, you may need to increase the number of PCR cycles to obtain sufficient yield after size selection Starting material should be in 1–20 µl of an alkaline, low ionic buffer such as the DNA Dilution Buffer (5 mM Tris-HCl pH 8.5) provided, or water. The optimal pH range of samples is 7.5–8.5. Avoid EDTA-containing buffers <br> ===A: Denaturation of DNA=== :- Aliquot appropriate amounts of DNA (100 pg to 10 ng) in a maximum volume of 20 µl in PCR tubes. :: I add 10ul of Dilution buffer to 10ul of eluted bisulfite converted DNA and transfer to 0.2mL reaction tube and spin down. :- Incubate the tubes at 94°C in a preheated, hot-lid thermal cycler '''(ChIP-94)''' for exactly 2 min :- Immediately remove the samples and place on ice for at least 2 minutes :- Spin the tubes briefly to bring down any condensation.<br> <br> === B: Dephosphorylation of 3’ Ends === :- Prepare enough Dephosphorylation Master Mix for all reactions, plus 10%, by combining the following reagents on cool box. ::{| {{table}} | style="width:240px;"|'''Components''' | style="width:100px;"|'''1x rxn''' | style="width:100px;"|'''4.4 rxn''' |- | DNA SMART Buffer (red cap)||3.25||14.30 |- | Shrimp Alkaline Phosphatase (red cap)||0.75||3.30 |- | Total volume per reaction||4.00|| |} '''NOTE:''' Add the Shrimp Alkaline Phosphatase to the buffer immediately prior to use. Mix well by gently vortexing and spin the tubes briefly in a microcentrifuge. Keep the DNA SMART Buffer on ice until its next use in Step V.D. :- Add 4 μl of the Dephosphorylation Master Mix to each reaction tube from Step V.A. Mix the contents of the tubes by gently vortexing, and spin the tubes briefly" :- Place the tubes in a preheated thermal cycler and run program ChIP-B. Leave the tubes at 4°C until the next step<br> :- '''ChIP-B:''' 37°C 10 min --> 65°C 5 min --> 4°C hold<br> '''NOTE:''' During the 65°C incubation step, prepare the T-Tailing Master Mix in Step V.C.1 below. <br> === C: T-Tailing === :- Prepare enough T-Tailing Master Mix for all reactions, plus 10%, by combining the following reagents on ice ::{| {{table}} | style="width:300px;"|'''Components''' | style="width:100px;"|'''1x rxn''' | style="width:100px;"|'''4.2 rxn''' |- | DNA SMART T-Tailing Mix (green cap)||1.00||4.20 |- | Terminal Deoxynucleotidyl Transferase (green cap)||1.00||4.20 |- | Total volume per reaction||2.00|| |} :- Add 2 μl of the T-Tailing Master Mix to each reaction tube. Mix the contents of the tubes by gently vortexing, and spin the tubes briefly" :- Place the tubes in a preheated thermal cycler and run program ChIP-C. Leave the tubes at 4°C until the next step. :- '''ChIP-C:''' 37°C 20 min --> 70°C 10 min -- > 4°C hold<br> <br> === D: Primer Annealing and Template Switching Reaction === The DNA SMART Poly(dA) Primer anneals to the ssDNA template followed by DNA replication and template switching. At the end of this step, the DNA is double stranded with partial adapters on both ends. '''NOTE:''' Prepare the Template Switching Master Mix (Step V.D.5) prior to starting the primer annealing step below. :- Primer Annealing: Add 2 μl of the DNA SMART Poly(dA) Primer (yellow cap) to each reaction tube. Mix the contents of the tubes by gently vortexing and spin the tubes briefly." :- Incubate the tubes at 94°C in a preheated, hot-lid thermal cycler (ChIP-94) for exactly 1 min. :- Immediately remove the samples and place on ice for at least 2 minutes. :- Spin the tubes briefly to bring down any condensation. '''NOTE:''' Proceed immediately to the next step :- Prepare enough Template Switching Master Mix for all reactions, plus 10%, by combining the following reagents on ice. ::{| {{table}} | style="width:300px;"|'''Components''' | style="width:100px;"|'''1rxn (ul)''' | style="width:100px;"|'''4.4 rxn''' |- | DNA SMART Buffer||6.00||26.40 |- | DNA SMART Oligonucleotide mix||6.00||26.40 |- | SMARTScribe Reverse Transcriptase||4.00||17.60 |- | Total||16.00|| |} '''Note:''' DO NOT add the SMARTScribe Reverse Transcriptase to the buffer until immediately prior to use. Mix well by gently vortexing and spin the tubes briefly in a micro centrifuge :- DNA Replication and Template Switching: Add the SMARTScribe Reverse Transcriptase (purple cap) to the Template Switching Master Mix prepared in Step V.D.5, then add 16 μl of the Template Switching Master Mix to each reaction tube. Mix the contents of the tubes by gently vortexing and spin the tubes briefly. :- Place the tubes in a preheated thermal cycler and run program ChIP-D. Leave the tubes at 4°C until the next step :- '''ChIP-D:''' 42°C 90 min --> 70°C 15 min -- > 4°C hold '''*Note:''' I left the reaction O/N at 4C before continuing to PCR. <br> === E. ChIP-Seq Library Amplification by PCR === == <span style="color:dodgerblue">RESULT TEST #1</span> == '''Note:''' I used realplex (Eppendorf) for amplification [[File:ZhangLab_2 2014-10-09 13hr 31min_1ng-15-20x-NTC15-18x.jpg| 500px]] [[File:ZhangLab_2 2014-10-09 14hr 11min_100pg-18-22x_NTC19-22.jpg| 500px]] [[File:ZhangLab_2 2014-10-09 13hr 40min_10pg-20-24x_NTC23-24x.jpg| 400px]] === Discussion (will be added) === <br> <br> ---- == <span style="color:limegreen"><u>TEST #2</u></span> == '''2014-10-09'''<br> * From previous experiment, I got amplified in NTC at the same position of the reaction with template DNA. Even the amplified level in NTC was lower than 1ng & 100pg input DNA, this seem to be very high. I did not see the different between 10pg input and NTC. * This might be contamination. I need to figure out where this signal from. * Actually, I should have included '''positive control''' using the fragmented DNA that came with the kit when I set up the reaction in step A: Dephosphorylation and I should also have set up another NTC in step A. This will help to identify that where did the contamination came from. It could be the step of MspI digestion and bisulfite conversion or since step A of the protocol. * This experiment, I will only use control fragmented DNA of the kit at the same amount of previous experiment and skip MspI and bisulfite conversion to make sure that the protocol works in my hands and see how NTC looks like when I skip the steps before. * I followed that protocol explained above from A to D. * The only difference is that I continue to step E (PCR) right after I finish step D. :- I initially dilute control fragmented DNA from 5ng/ul to 0.5ng/ul or 500pg/ul. In this step, I did not confirm the concentration of control DNA as I usually did. ::{| {{table}} border = 1 | align="center" style="width:120px;"|'''Conc. (pg/ul)''' | align="center" style="width:80px;"|'''500''' | align="center" style="width:80px;"|'''50''' | align="center" style="width:80px;"|'''5''' |- | Stock used (pg/ul)||5000.00||500.00||50.00 |- | Volume of stock (ul)||2.00||10.00||10.00 |- | EB (ul)||18.00||90.00||90.00 |- | Total||20.00||100.00||100.00 |} ===A: Denaturation of DNA=== :- Aliquot appropriate amounts of DNA (100 pg to 10 ng) in a maximum volume of 20 µl in PCR tubes. :: I add 18ul of Dilution buffer to each 0.2mL strip tube and add 2ul diluted control DNA, mix well and spin down. :- Followed protocol describe above for A. <br> === B: Dephosphorylation of 3’ Ends === :- Followed protocol describe above for B. ::::{| {{table}} | style="width:240px;"|'''Components''' | style="width:100px;"|'''1x rxn''' | style="width:100px;"|'''4.4 rxn''' |- | DNA SMART Buffer (red cap)||3.25||14.30 |- | Shrimp Alkaline Phosphatase (red cap)||0.75||3.30 |- | Total volume per reaction||4.00|| |} :- Add 4 μl of the Dephosphorylation Master Mix to each reaction tube from Step V.A. Mix the contents of the tubes by gently vortexing, and spin the tubes briefly" :- Place the tubes in a preheated thermal cycler and run program ChIP-B. Leave the tubes at 4°C until the next step<br> :- '''ChIP-B:''' 37°C 10 min --> 65°C 5 min --> 4°C hold<br> '''NOTE:''' During the 65°C incubation step, prepare the T-Tailing Master Mix in Step V.C.1 below. <br> === C: T-Tailing === :- Followed protocol describe above for C. ::{| {{table}} | style="width:300px;"|'''Components''' | style="width:100px;"|'''1x rxn''' | style="width:100px;"|'''4.2 rxn''' |- | DNA SMART T-Tailing Mix (green cap)||1.00||4.20 |- | Terminal Deoxynucleotidyl Transferase (green cap)||1.00||4.20 |- | Total volume per reaction||2.00|| |} :- Add 2 μl of the T-Tailing Master Mix to each reaction tube. Mix the contents of the tubes by gently vortexing, and spin the tubes briefly" :- Place the tubes in a preheated thermal cycler and run program ChIP-C. Leave the tubes at 4°C until the next step. :- '''ChIP-C:''' 37°C 20 min --> 70°C 10 min -- > 4°C hold<br> <br> === D: Primer Annealing and Template Switching Reaction === :- Followed protocol describe above for D. :- Primer Annealing: Add 2 μl of the DNA SMART Poly(dA) Primer (yellow cap) to each reaction tube. Mix the contents of the tubes by gently vortexing and spin the tubes briefly." :- Incubate the tubes at 94°C in a preheated, hot-lid thermal cycler (ChIP-94) for exactly 1 min. :- Immediately remove the samples and place on ice for at least 2 minutes. :- Spin the tubes briefly to bring down any condensation. '''NOTE:''' Proceed immediately to the next step ::{| {{table}} | style="width:300px;"|'''Components''' | style="width:100px;"|'''1rxn (ul)''' | style="width:100px;"|'''4.4 rxn''' |- | DNA SMART Buffer||6.00||26.40 |- | DNA SMART Oligonucleotide mix||6.00||26.40 |- | SMARTScribe Reverse Transcriptase||4.00||17.60 |- | Total||16.00|| |} :- DNA Replication and Template Switching: Add the SMARTScribe Reverse Transcriptase (purple cap) to the Template Switching Master Mix prepared in Step V.D.5, then add 16 μl of the Template Switching Master Mix to each reaction tube. Mix the contents of the tubes by gently vortexing and spin the tubes briefly. :- Place the tubes in a preheated thermal cycler and run program ChIP-D. Leave the tubes at 4°C until the next step :- '''ChIP-D:''' 42°C 90 min --> 70°C 15 min -- > 4°C hold '''*Note:''' I continue to amplification by PCR step right after I finish step D.<br> <br> === E. ChIP-Seq Library Amplification by PCR === :- I used half of the template for PCR in total 50ul. ::{| {{table}} | style="width:240px;"|'''Components''' | style="width:80px;"|'''Total 100ul''' | style="width:80px;0f0f0;"|'''Total 50ul''' | style="width:80px;"|'''4.5 rxn''' |- | SeqAmp PCR Buffer (2X)||50.00||25.00||112.5 |- | Forward PCR Primer (12.5uM)||2.00||1.00||4.5 |- | Reverse PCR Primer (12.5uM, R2)||2.00||1.00||4.5 |- | 50X SYBR||0.80||0.40||1.8 |- | SeqAmp DNA Polymerase||2.00||1.00||4.5 |- | Total||56.80||28.40|| |} :- Aliquot 28.4ul of PCR mix and add 22ul of template. :- I then split them in 8ul x 6 to vary PCR cycle number for 15-20 cycles<br> <br> :- '''ChIP-PCR:''' 94C 1min --> 15-20X [98C 15s -> 55C 15s -> 68C 30s] --> 10C hold<br> <br> :- Add 2ul of 6X loading dye to 8ul of PCR product. :- Loaded 6ul of PCR-dye mix in 6% TBE gel == <span style="color:limegreen">RESULT TEST #2</span> == '''Note:''' I used realplex (Eppendorf) for amplification [[File:ZhangLab_2 2014-10-09 20hr 37min_DNASMART-controlDNA_15-16x.jpg| 500px]] [[File:ZhangLab_2 2014-10-09 20hr 37min_DNASMART-controlDNA_17-18x.jpg| 500px]] [[File:ZhangLab_2 2014-10-09 20hr 37min_DNASMART-controlDNA_19-20x.jpg| 500px]] ---- == <span style="color:Crimson"><u>TEST #3</u></span> == '''2014-10-13'''<br> * I want to test where is the strong background in NTC (likely to be contamination) from TEST #1 came from. Also I want to test how is the result if I do bisulfite conversion on control fragmented DNA. Would I still see a high background in NTC? If yes, I might say contamination derived from bisulfite conversion step. * The last thing I want to test is that doing bisulfite conversion on gDNA (CANC1 samples) without MspI will generate small fragment DNA enough for cDNA synthesis? * In this experiment I included below sample amount 100pg, ** MspI --> BIS-CVT *** CANC1 *** NTC ** BIS-CVT (no MspI) *** CANC1 *** NTC *** Control fragmented DNA *** NTC ** STD protocol without MspI nor BIS-CVT *** Control fragmented DNA *** NTC * All diluted DNAs were prepared in TEST #1 and TEST #2 experiments. === <u>MspI reaction Mix</u> === ::{| {{table}} | style="width:180px;"|'''Components''' | style="width:100px;"|'''1 rxn''' | style="width:100px;"|'''2,2 rxn''' |- | MspI (10U/ul)||1.00||2.20 |- | 10x Tango Buffer||2.00||4.40 |- | H2O||7.00||15.40 |- | Total||10.00|| |} === Bisulfite conversion === * Sample, ** MspI --> BIS-CVT *** CANC1 --> add 130ul Lightning conversion reagent directly to the tube *** NTC --> add 130ul Lightning conversion reagent directly to the tube ** BIS-CVT (no MspI) *** CANC1: Mix 10ul of 10pg/ul CANC1 & 10ul H2O --> add 130ul Lightning conversion reagent *** NTC: Add 20ul H2O --> add 130ul Lightning conversion reagent *** Control fragmented DNA: Mix 2ul of 50pg/ul control fragmented DNA and 18ul H2O --> add 130ul Lightning conversion reagent *** NTC: Add 20ul H2O --> add 130ul Lightning conversion reagent :- Perform bisulfite conversion by EZ-Lightning DNA Methylation kit using instructions provided by manufacturer without using tRNA as carrier. :- Elute with 11.5ul of Elution Buffer. The left over volume of DNA is ~10ul. :- Continue to the the steps of DNA SMART ChIP-Seq protocol ===A: Denaturation of DNA=== will be added == <span style="color:Crimson">RESULT TEST #3</span> == {| {{table}} border = 1 | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total 100ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total 40ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''8.5 rxn''' |- | SeqAmp PCR Buffer (2X)||50.00||20.00||170.00 |- | Forward PCR Primer (12.5uM)||2.00||0.80||6.80 |- | Reverse PCR Primer (12.5uM, R2)||2.00||0.80||6.80 |- | 50X SYBR||0.80||0.32||2.72 |- | SeqAmp DNA Polymerase||2.00||0.80||6.80 |- | Total||56.80||22.72|| |} :- Aliquot 22.72ul + DNA template 17.60ul :- '''ChIP-PCR:''' 94C, 1min -->[98C, 15s -> 55C, 15s, 68C, 30s] 18X --> 4C hold '''Note:''' I used Chromo4 (BIO-RAD) for amplification<br> '''qPCR of the reaction with and without bisulfite conversion''' [[File:141014_TEST3_control.PNG| 400px]] [[File:141014_TEST3_plus-bis-cvt.PNG| 400px]] g = gDNA of cancer cell line f = control fragmented DNA '''PAGE verification''' [[File:ZhangLab_2 2014-10-14 12hr 44min_TEST3_18x.jpg| 500px]] === WHAT TO DO NEXT === * According to the contamination, I am pretty sure that it came from bisulfite conversion step as I did not had contamination from MspI digestion from previous experiments, and I used all new clean reagents for MspI digestion. The sources could be either reagents or during centrifugation especially during elution step as the 1.5mL collection tubes were opened. I UV all regents for bisulfite conversion and column/tube except bisulfite conversion reagent that needed to be protected from light. It's hard to say what is the exact source of contamination. The level of contamination are equal in all samples and NTC, so it's likely that it might come from reagent contamination. * I'm planning to repeat the experiment by trying bisulfite conversion kit using bead-base in stead of column-base kit to avoid contamination from centrifugation step. * Another thing that I need to be concerned is how to get it work for 10pg input. With the standard protocol, I could not see amplification of 10pg reaction. If I need to get the methylation experiment works, I need to get the standard 10pg reaction work first. * I ordered the bisulfite conversion kit, "BisulFlash DNA Bisulfite Conversion Mag-96 Kit", from Epigentak company. I have never tried the kit from this company. I am interested to use this kit because every step was performed in 200ul strip tube and no need for centrifugation. Zymo also has the bead kit too, but there is some step that need to heat on the themomixer with the open deep well plate. This might cause contamination. In addition, the working volume of Zymo kit is very high, which may not be suitable for the very low input reaction <100pg reaction. For the kit from Epigentak, I need to verify the conversion efficiency and recovery rate compare to Zymo kit too.
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