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==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
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