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===oxbs fragmentation/degradation test 2=== * Previous test used EZ-Methylation Lightning Kit, this test using Epitect Bisulfite Plus Kit * Performed oxBS, without Bio-Rad P6 columns (test if column is necessary after QIAEX II purification) ** A. 22.75 uL QIAEX II purified amplicons and 1.25 uL 1M NaOH - 37C for 40 minutes in ThermoMixer ** B. 9.1 uL QIAEX II purified amplicons and 0.5 1 M NaOH (for BS control) - 37C for 1 hour in Thermocycler ** added 1 uL 15 mM KRuO4 to (A) and incubate for 1 hour at 40C. ** used all of (B) for bisulfite conversion in 1 reaction set up ** separated 9.8 uL of (A) for 1 reaction and rest of (A) in another bisulfite reaction * QUBIT {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Treatment''' | align="center" style="background:#f0f0f0;"|'''Qubit concentration''' | align="center" style="background:#f0f0f0;"|'''Units''' | align="center" style="background:#f0f0f0;"|'''Dilution''' | align="center" style="background:#f0f0f0;"|'''Sample concentration''' | align="center" style="background:#f0f0f0;"|'''Units''' |- | B||bs||150.00||ng/mL||200||30||ng/uL |- | B||ox||153.00||ng/mL||200||30.6||ng/uL |- | B||ox2||280.00||ng/mL||200||56||ng/uL |- | E||bs||376.00||ng/mL||200||75.2||ng/uL |- | E||ox||421.00||ng/mL||200||84.2||ng/uL |- | E||ox2||646.00||ng/mL||200||129.2||ng/uL |- | F||bs||74.70||ng/mL||200||14.94||ng/uL |- | F||ox||69.80||ng/mL||200||13.96||ng/uL |- | F||ox2||130.00||ng/mL||200||26||ng/uL |} {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Concentration(ng/uL)''' | align="center" style="background:#f0f0f0;"|'''DNA volume''' | align="center" style="background:#f0f0f0;"|'''Mass (ng)''' | align="center" style="background:#f0f0f0;"|'''10 mM dNTPs''' | align="center" style="background:#f0f0f0;"|'''10X ThermoPol Buffer''' | align="center" style="background:#f0f0f0;"|'''100X NAD+''' | align="center" style="background:#f0f0f0;"|'''PreCR Repair Mix''' | align="center" style="background:#f0f0f0;"|'''Water''' |- | bs-B||30||3.333||100||0.25||2.5||0.25||0.5||18.17 |- | bs-E||75.2||1.330||100||0.25||2.5||0.25||0.5||20.17 |- | bs-F||14.94||6.693||100||0.25||2.5||0.25||0.5||14.81 |- | oxBS-B||30.6||3.268||100||0.25||2.5||0.25||0.5||18.23 |- | oxBS-E||84.2||1.188||100||0.25||2.5||0.25||0.5||20.31 |- | oxBS-F||13.96||7.163||100||0.25||2.5||0.25||0.5||14.34 |- | bs-B/-||30||3.333||100||0.25||2.5||0.25||0||18.67 |- | bs-E/-||75.2||1.330||100||0.25||2.5||0.25||0||20.67 |- | bs-F/-||14.94||6.693||100||0.25||2.5||0.25||0||15.31 |- | oxBS-B/-||30.6||3.268||100||0.25||2.5||0.25||0||18.73 |- | oxBS-E/-||84.2||1.188||100||0.25||2.5||0.25||0||20.81 |- | oxBS-F/-||13.96||7.163||100||0.25||2.5||0.25||0||14.84 |} * PreCR repair at 37C for 2 hours. * Real time PCR with Phusion U {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''Concentration''' | align="center" style="background:#f0f0f0;"|'''1X volume''' | align="center" style="background:#f0f0f0;"|'''Final concentration''' | align="center" style="background:#f0f0f0;"|'''6.2X''' |- | Nuclease free water||NA||33.65||NA||208.63 |- | Phusion HF Buffer||10X||10||1X||62 |- | Primer F/R (B/E/F)||10 uM||1.5||300 nM||9.3 |- | dNTP||10 mM||1.25||250 nM||7.75 |- | Phusion U||2U/uL||0.8||1.6 U||4.96 |- | Sybr Green||50X||0.8||0.8X||4.96 |- | Template||NA||2||NA||NA |} * Real time PCR curves ** E > F > B (5hmC amplified the slowest, none or small difference between oxBS and BS). NOTE: same amount of DNA used repair & no repair control reactions. ** PreCR enzymes treatment degraded the DNA as expected. ** Why does BS and oxBS have very similar outcomes? If oxidized DNA have more problem amplifying then it should amplify slower than bisulfite alone! [[File:control_dna_v3_test_postPreCR_repair_curves_test2.png | 300px]] * Ran PAGE gel to confirm the correct size amplicons were generated. * Pool B/E/F for the next tests: since B/E/F can be amplified out, I pooled equal molar ratio of each amplicons. ** 200 ng B, 277.2 ng E, 293.1 ng F {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Pooled sample ID''' | align="center" style="background:#f0f0f0;"|'''B''' | align="center" style="background:#f0f0f0;"|'''E''' | align="center" style="background:#f0f0f0;"|'''F''' | align="center" style="background:#f0f0f0;"|'''Water''' |- | #1 - BS, 0.1X||4.85||5.12||14.3||5.72 |- | #2 - oxBS, 0.1X||5.39||4.36||19.67||0.58 |- | #3 - BS, 0.2X repair||6.85||6.96||0||16.19 |- | #4 - oxBS, 0.2X repair||9.71||10.83||0||9.46 |- | #5 - BS, 0.2X no repair||5.54||4.07||11.82||8.57 |- | #6 - oxBS, 0.2X no repair||4.83||4.13||12.32||8.72 |} * Digestion test {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Reaction''' | align="center" style="background:#f0f0f0;"|'''Enzyme''' | align="center" style="background:#f0f0f0;"|'''Enzyme volume''' | align="center" style="background:#f0f0f0;"|'''10X NEB2.1 buffer''' | align="center" style="background:#f0f0f0;"|'''Water''' | align="center" style="background:#f0f0f0;"|'''Pooled amplicons''' |- | Pool #X||SphI||1||1||6||2 |- | Pool #X||no SphI||0||1||7||2 |} * qPCR to calculate conversion efficiencies {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''Concentration''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''Final concentration''' | align="center" style="background:#f0f0f0;"|'''24.5X volume''' |- | KAPA Sybr Fast qPCR Master Mix||2X||5||1X||122.5 |- | B/E/F primers||10 uM||0.3||300 nM||7.35 |- | Template (0.1X, 0.01X)||NA||4.7||NA||--- |} * Digestion test results: Expectations: B amplicons should be digested in pool #1, #3, #5, should not be digested in #2, #4, #6 E amplicons should be digested in EVERY POOL. F amplicons should not be digested in EVERY POOL. Results: B amplicons 0% digested (1), 0% digested (2), 0% digested (3), 14-22% digested (4), 0-14% digested (5), 0-20% digested (6). E amplicons 88-95% digested (1), 88-95% digested (2), 72-89% digested (3), 71-91% digested (4), 89-94% digested (5), 88-94% digested (6) F amplicons was not digested in every pool * E/F amplicons digestion were expected, except for digestion efficiency it seems for E amplicons * B amplicons digestion was expected in (2), and perhaps show poor conversion because (4) and (6) showed digestion. But (1), (3), and (5) also showed low digestion, which meant that these positions did not contain C (before or after bisulfite conversion?) * Prepare Phusion amplicons for sequencing: ** Using JumpStart Taq polymerase to add A tails - 70C for 40 minutes ** Purify using 2X AMPure beads. Elute with 20 uL water. Keep beads! ** T4 PNK to phosphorylate 5' ends: +2.5 uL T4 PNK +2.5 uL T4 DNA ligation buffer: 37C for 40 minutes ** Add adaptors (ligation overnight): +1 uL T4 DNA ligation buffer, +1 T4 DNA ligase (6U), +1 TruSeq adaptors, +7 uL water: 16C overnight. * Pooled samples + adaptor IDs #1 TS 15 #2 TS 16 #3 TS 27 #4 TS 18 #5 TS 19 #6 TS 20 {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''Concentration''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''Final concentration''' | align="center" style="background:#f0f0f0;"|'''6.2X''' |- | Phusion U amplicons||NA||25||NA||NA |- | PCR Buffer||10X||3.5||1X||21.7 |- | MgCl2||25 mM||2.1||1.5 mM||13.02 |- | dATP ||10 mM||0.7||0.2 mM||4.34 |- | JumpStart Taq Pol||2.5 U/uL||1||2.5 U||6.2 |- | Water||NA||2.7||NA||16.74 |} * Purify ligation products with AMPure beads * Nanodrop Pool #1: 26.0 ng/uL Pool #2: 25.2 ng/uL Pool #3: 18.2 ng/uL Pool #4: 22.4 ng/uL Pool #5: 31.1 ng/uL Pool #6: 30.5 ng/uL * Test qPCR {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''Concentration''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''Final concentration''' | align="center" style="background:#f0f0f0;"|'''16.5X volume''' |- | KAPA Sybr Fast qPCR Master Mix||2X||5||1X||31 |- | Primer F/R (TruSeq)||10 uM||0.3||300 nM||1.86 |- | Nuclease free water||NA||3.7||NA||22.94 |- | Template||NA||1||NA||--- |} * Full qPCR - 6 cycles {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''Concentration''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''Final concentration''' | align="center" style="background:#f0f0f0;"|'''16.5X volume''' |- | KAPA Sybr Fast qPCR Master Mix||2X||50||1X||310 |- | Primer F/R (TruSeq)||10 uM||3||300 nM||18.6 |- | Nuclease free water||NA||37||NA||229.4 |- | Template||NA||10||NA||--- |} * Purified with Bioneer columns and Nanodrop: #1 70.7 ng/uL #2 36.4 ng/uL #3 33.2 ng/uL #4 49.7 ng/uL #5 64.9 ng/uL #6 42.6 ng/uL * Pooled 100 ng of each together to make 1 big pool * Purified big pool with AMPure beads.
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