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= Repeating of BIS/DNA SMART ChIP-Seq protocol = [[noi:DMR220k_LabNotes#2014|'''Back to calendar''']] * According to experiment on 2014-10-29, I still see amplification of contaminants in BIS-NTC. In that experiment, I used ''BisulFlash DNA Methylation kit (bead-base)'' from Epigentek. For this experiment, I would ignore a bisulfite conversion rate and recovery yield of this kit. I just wanted to try the new kit with the price is affordable to see whether I can remove contamination. Even the contamination was observed, the level was lower than Zymo DNA Methylation-Lightning kit (column-based). * In this experiment, I want to repeat experiment again using Zymo DNA Methylation-Lightning Magprep kit. I want to see if the level of contamination is comparable to the first experiment that I tried with DNA Methylation-Lightning column-based kit in the first experiment on 2014-10-08. This might explain about contamination in CT conversion reagent and the procedures between column and bead. * I also wanted to compare the yield after amplification between BIS-100ng input and BIS-NTC. * I used the old "Zymo DNA Methylation-Lightning Magprep kit" I purchased in September 2013 as getting the new kit is too expensive for trial experiment. However, I believe that the reagents should be clean. I UV all reagent except conversion reagent. * I used ClonTech control fragmented human gDNA for this experiment. I followed the procedures of bisulfite conversion by zymo DNA Methylation-Lightning Magprep kit and resuspend with 22ul Elution buffer. Then transfer 20ul of converted DNA for library preparation * I used the same procedure of library preparation using ClonTech DNA SMRT-Seq kit as describe in [[Noi/NOTES/2014-10-29#A:_Denaturation_of_DNA|'''2014-10-29''']] == DNA SMRT ChIP-Seq library preparation == ===A: Denaturation of DNA=== {| {{table}} border = 1 | align="center" style="width:200px;background:#f0f0f0;"|'''Template''' | align="center" style="width:80px;background:#f0f0f0;"|'''Input amount''' | align="center" style="width:80px;background:#f0f0f0;"|'''BIS-CVT''' | align="center" style="width:80px;background:#f0f0f0;"|'''Stock conc.''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Dilution buffer''' |- | Control fragmented DNA||1ng||YES||NA||20.00||0.00 |- | Control fragmented DNA||100pg||YES||NA||20.00||0.00 |- | NTC||NA||YES||NA||20.00||0.00 |- | NTC||NA||YES||NA||20.00||0.00 |- | Control fragmented DNA||100pg||no||50pg/ul||2.00||18.00 |- | NTC||NA||no||NA||0.00||20.00 |} :- 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 6.2 rxn Dephosphorylation Master Mix for all reactions by combining the following reagents on ice. ::{| {{table}} | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:100px;background:#f0f0f0;"|'''1rxn (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''6.2 rxn''' |- | DNA SMART Buffer||3.25||20.15 |- | Shrimp Alkaline Phosphatase ||0.75||4.65 |- | Total||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 7.2rxn T-Tailing Master Mix for all reactions by combining the following reagents on ice ::{| {{table}} | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:100px;background:#f0f0f0;"|'''1rxn (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''6.2 rxn''' |- | DNA SMART T-Tailing Mix||1.00||6.2 |- | Terminal Deoxynucleotidyl Transferase||1.00||6.2 |- | Total||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 6.2rxn Template Switching Master Mix for all reactions by combining the following reagents on ice. ::{| {{table}} | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:100px;background:#f0f0f0;"|'''1rxn (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''6.2 rxn''' |- | DNA SMART Buffer||6.00||37.20 |- | DNA SMART Oligonucleotide mix||6.00||37.20 |- | SMARTScribe Reverse Transcriptase||4.00||24.80 |- | 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 === * I would initially do a quick qPCR to test the PCR cycle number ::{| {{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 15ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''6.5 rxn''' |- | SeqAmp PCR Buffer (2X)||50.00||7.50||48.75 |- | Forward PCR Primer (12.5uM)||2.00||0.30||1.95 |- | Reverse PCR Primer (12.5uM, R3)||2.00||0.30||1.95 |- | 50X SYBR||0.40||0.06||0.39 |- | SeqAmp DNA Polymerase||2.00||0.30||1.95 |- | Total||56.40||8.46|| |} :- Aliquot 8.46ul + 6.6ul of DNA template :- Split 4.5ul to 3X 8-tube strip :- '''ChIP-PCR:''' 94C, 1min -->[98C, 15s -> 55C, 15s, 68C, 30s] X16, 18 19 --> 4C hold '''Note:''' I used REalplex (Eppendorf) for amplification<br> :- Load all 4.5ul PCR product in 6% TBE gel. * Note: I did 19 cycles because I wanted to see the background of regular reaction (-BIS) when I ran PCR over 18 cycles. [[File:ZhangLab_2 2014-10-31 18hr 58min_LightningMagprep-DNASMRT-16X.jpg| 400px]] [[File:ZhangLab_2 2014-10-31 18hr 58min_LightningMagprep-DNASMRT-18X.jpg| 405px]] [[File:ZhangLab_2 2014-10-31 18hr 58min_LightningMagprep-DNASMRT-19X.jpg|400px]] * From the gel image above, I can observe amplification in +bis/NTC. The level looked lower than +bis/100pg and +bis/1ng input. at 16 and 18 cycles. For 19 cycles, the amplification was getting to saturated. * No amplification in -bis/NTC was observed at 18 cycles, suggesting no contamination during library prep procedures. * At the same input between +bis/100pg and -bis/100pg, the intensity of PCR product from converted DNA no converted DNA. This should be explained by the contamination in +bis/100pg. * I just got response email from Nathalie, the one who developed the template switching protocol. She suggest to compare the yield after PCR between the reaction with different amount of bis-DNA and bis-NTC by Qubit or Bioanalyzer quantification. * I then amplified the left over reversed transcribed DNA template with indexed primers (just in case, we need to verify by sequencing) using the same condition above at 17 cycles. ::{| {{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 85ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''6.5 rxn''' |- | SeqAmp PCR Buffer (2X)||50.00||42.50||276.25 |- | Forward PCR Primer (12.5uM, F2)||2.00||1.70||11.05 |- | Reverse PCR Primer (12.5uM, RXX)||2.00||1.70||0.00 |- | 50X SYBR||0.40||0.34||2.21 |- | SeqAmp DNA Polymerase||2.00||1.70||11.05 |- | Total||56.40||47.94|| |} :- Aliquot 46.24ul to each DNA template tube containing 37.4ul DNA template + 1.7ul of Reverse PCR primer ==== Illumina indexing ==== '''CT:''' ClonTech control fragmented human gDNA :{| {{table}} border= 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Template''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' |- | BIS-CT 1ng||R1 |- | BIS-CT 100pg||R2 |- | BIS-NTC1||R4 |- | BIS-NTC2||R5 |- | CT-100pg||R6 |- | NTC||R7 |} *- Mix well :- Split 42ul to 2X 8-tube strip :- '''ChIP-PCR:''' 94C, 1min -->[98C, 15s -> 55C, 15s, 68C, 30s] X17 --> 4C hold :- I did 1 round of AMPure bead purification using 1X AMPure bead. The protocol suggested to use 0.9X to remove all fragment below 250bp. I saw the amplicon size close to 200bp, so I use 1X volume. :- I did not use resuspension buffer provided with the kit, but use EB buffer to elute (25ul). It's quite difficult to to resuspend the beads since the component of PCR mix is quite sticky. In the future , I might try using library resuspension buffer from the kit. :- Measure concentration of each sequencing library by Qubit dsDNA HS assay (2ul for assay) and calculate the yields. === Qubit dsDNA HS assay result and yield comparison === :{| {{table}} border = 1 | align="center" style="width:140px;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)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Yield in 22ul (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Yield after background subtraction (ng)''' |- | BIS-CT 1ng||70.8||ng/mL||2||100||7.08||155.76||146.85 |- | BIS-CT 100pg||7.94||ng/mL||2||100||0.79||17.468||8.558 |- | BIS-NTC1||4.12||ng/mL||2||100||0.41||9.064|| |- | BIS-NTC2||3.98||ng/mL||2||100||0.4||8.756|| |- | CT-100pg||5.12||ng/mL||2||100||0.51||11.264|| |- | NTC||Out Of Range||<50ng/mL||2||100||NA||NA|| |} :- Average yield of +bis/NTC = 8.91ng :- Subtract the background :: +bis/1ng - +bis/NTC = 155.76 - 8.91 = 145.85ng :: +bis/100pg/NTC - +bis/NTC = 17.468 - 8.91 = 8.558ng :- Compare the yield between +bis/1ng and +bis/100pg = 145.85ng: 8.558 ng --> 17X. :- For +bis/100pg, there should be the amount contaminants in the library ~50%. This should be concerned if wasting 50% of sequencing amount for sequencing contamination is affordable. :- For +bis/10pg input, assume the yield ~20 time less than +bis/100pg input = 8.558/20 = ~0.4279ng. This amount is less than 5% of non-contaminant DNA in the library. This contamination level should not be acceptable for 10pg input. ==== Dr. Zhang opinion? ==== * Should we do sequencing on these libraries to see the contamination sources? -> from human, bacteria or other organisms? * How to resolve contamination issue from bisulfite conversion. * The major goal of using this protocol, for regular library prep or bisulfite sequencing library prep? The sensitivity, for low input or single cell level that we want to achieve? * The acceptable level of contamination in 100pg or 10pg input?
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