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AlanFung:LabNotes/CTCF/2011-7-12
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==Improved protocol for Identifying source of loss during Nextera Bisulfite conversion with 1ng gDNA== ===Objective=== #Want to figure out the source of loss during Nextera Bisulfite conversion #Use qPCR to identify the source of the loss #Eliminate source of contamination ===Samples=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Protocol''' | align="center" style="background:#f0f0f0;"|'''1''' | align="center" style="background:#f0f0f0;"|'''2''' | align="center" style="background:#f0f0f0;"|'''3''' | align="center" style="background:#f0f0f0;"|'''4''' | align="center" style="background:#f0f0f0;"|'''5''' | align="center" style="background:#f0f0f0;"|'''6''' | align="center" style="background:#f0f0f0;"|'''7''' |- | Low-Input Nextera||X||X||X||X||X||X||X |- | Protease Digestion (1:100 Dilution)||X||X||X||X||X||X||X |- | Klenow Extension||X||X||X||X||X||X||X |- | Heat||||X||X||X|||||| |- | Heat w bisulfite||||||||||X||||X |- | Desulphonation||||||||X||X||||X |- | Column||||||X||X||X||||X |- | Amplify||X||X||X||X||X||X||X |} #Nextera Low-Input, Normal Shendure Protocol #Nextera Low-Input, Heat #Nextera Low-Input, Heat, Column Purification #Nextera Low-Input, Heat, Desulphonate, and Column Purify #Nextera Low-Input, Heat, Bisulfite, Desulphonate, and Column Purify #NTC1 - normal shendure protocol #NTC2 -shendure + bisulfite protocol * "Nextera Low-Input": Perform Shendure Protocol, Protease Treat with 1/100 Dilution, Klenow Extend ** Used 1 uL of 1 ng/uL Jurkat DNA in first five tubes, 1 uL H2O in NTC * '''Normal Protocol:''' Amplify using KapaKlenow Protocol * '''Heat:''' After heat, amplify using KapaKlenow Protocol * '''Heat/Column:''' After heat, purify through Zymo column, skipping desulphonation step; amplify using KapaKlenow Protocol * '''Heat/Desulphonate/Column:''' After heat, purify through Zymo column with desulphonation step; amplify using KapaKlenow Protocol * '''Heat/Bisulfite/Desulphonate/Column:''' After heating in bisulfite solution, purify through Zymo column with desulphonation step; amplify using KapaKlenow protocol * '''NTC1:''' Just do normal protocol, but with water instead of 1 ng DNA * '''NTC2:''' repeat shendure protocol and bisulfite conversion with NTC ==Protocol== ===Elimination of source of contamination=== *Clean with EtOH and DNAse wash hood and UV fo 15mins *Use newly opened tips *UV all tubes *UV RT-PCR Grade water, EB Buffer and Protease ===Low-Input Tagmentation with Nextera=== * Add 1 uL of 1:50 Diluted Nextera Transposase, 1 uL Nextera 5x HMW Buffer, and 1 uL H2O to 2ul GM12878 gDNA (0.5ng/ul) *Sample 1-5 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | 1:50 Nextera Transposase||1||5 |- | 5X Nextera HMW Buffer||1||5 |- | RT-PCR Water||1||5 |- | 0.5ng/ul gDNA||2||10 |- | Total||5||25 |} *Sample 6-7 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | 1:50 Nextera Transposase||1||5 |- | 5X Nextera HMW Buffer||1||5 |- | RT-PCR Water||3||15 |- | 0.5ng/ul gDNA||0||0 |- | Total||5||25 |} * Incubate at 55 C for 5 minutes ===Protease Digestion=== * Add 1 uL of 1:100 diluted Qiagen Protease (5 AU/mL, diluted to 0.05 AU/mL) to solution *Add 5ul to sample 1-5 *Add 2 ul to sample 6-7 * Incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes ===Klenow Extension=== * Add 1 uL of 10 U/uL Klenow exo-minus DNA Polymerase and 1 uL of 1 mM Nucleotide Mix (dATP, dGTP, m-dCTP, dTTP) to solution *Add 5ul klenow and 5ul dNTP to sample 1-5 *Add 2 ul klenow and 2 ul dNTP to sample 6-7 * Incubate at 30 C for 30 minutes *Aliquot 8ul to each tubes *keep sample 1 & 6 at 4C for later amplification ===Bisulfite Conversion=== * Add 130 uL of CT Conversion Reagent (Zymo Bisulfite direct Kit) to sample 5 and 7 * Incubate at 98 C for 8 min, 64 C for 3.5 hours; hold at 4 C when finished *Incubate sample 2, 3, and 4 at a separate block for it has less volume ===Column Purification with/without Desulphonation=== *4, 5 and 7 desulphonation sample 3 no desulphonation *Set sample 2 aside until amplification * Follow instructions in Zymo Bisulfite Direct kit for purification * Skip desulphonation step when necessary ===Amplification with Kapa and Klenow=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | DNA||10||Do not add to MM |- | H2O||12||92.4 |- | 10uM Orange||1||7.7 |- | 10uM Blue||1||7.7 |- | 10U/ul Klenow Exo -||1||7.7 |- | 2X Kapa||25||192.5 |- | Total||50||308 |} * KapaKlenow uses Klenow for 2nd strand synthesis, Kapa for PCR * Add 1 uL Orange Primer, 1 uL Blue Primer, 25 uL Kapa SYBR Supermix, and 1 uL Klenow exo-minus (10 U/uL) to solution * Perform PCR using the following thermocycler settings: 30 C, 10 minutes 72 C, 3 minutes 95 C, 30 seconds == 95 C, 10 seconds 58 C, 30 seconds 72 C, 3 minutes == x 40 cycles 72 C 10 minutes Hold at 4 C ==Results== [[File:07_13_11_qpcr.jpg|900px]] {| {{table}} | align="center" style="background:#f0f0f0;"|'''Protocol''' | align="center" style="background:#f0f0f0;"|'''1''' | align="center" style="background:#f0f0f0;"|'''2''' | align="center" style="background:#f0f0f0;"|'''3''' | align="center" style="background:#f0f0f0;"|'''4''' | align="center" style="background:#f0f0f0;"|'''5''' | align="center" style="background:#f0f0f0;"|'''6''' | align="center" style="background:#f0f0f0;"|'''7''' |- | Low-Input Nextera||X||X||X||X||X||X||X |- | Protease Digestion (1:100 Dilution)||X||X||X||X||X||X||X |- | Klenow Extension||X||X||X||X||X||X||X |- | Heat||||X||X||X|||||| |- | Heat w bisulfite||||||||||X||||X |- | Desulphonation||||||||X||X||||X |- | Column||||||X||X||X||||X |- | Amplify||X||X||X||X||X||X||X |} #Nextera Low-Input, Normal Shendure Protocol #Nextera Low-Input, Heat #Nextera Low-Input, Heat, Column Purification #Nextera Low-Input, Heat, Desulphonate, and Column Purify #Nextera Low-Input, Heat, Bisulfite, Desulphonate, and Column Purify #NTC1 - normal shendure protocol #NTC2 -shendure + bisulfite protocol *Results show that heat and bisulfite conversion step (sulphonation or deamination?) cause the most lost during bisulfite conversion *#6 NTC shoots up earlier than #5, which is weird. It was better than the last experiment where we saw NTC shoots up first. I say the extra sterilization steps cuts down the contamination but didn't completely eliminate the source. *About 5 cycle difference when comparing library with and without bisulfite conversion, around 30 fold difference. ==To Do== *I guess the epigentek Bisulflash DNA modification kit will be a good candidate to cut down the DNA lost during bisulfite conversion, since the conversion time is cut down to 20 minutes
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