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AlanFung:LabNotes/CTCF/2011-6-30
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==N9-MDA Bisulfite Converted Sequencing Library Construction == ==Objective== *Figure out source of contamination from 6/28 experiment *Repeat 6/28 experiment with only gDNA (UV EB buffer, use RT-PCR grade water as NTC) *Sample List #A-600pg gDNA W 0.1ul proteinase K #B-600pg gDNA W proteinase k #C-600pg gDNA W/O proteinase k #D-600pg gDNA W protease #E-NTC W proteinase K #F-NTC W/O proteinase K ==Overview== #Proteinase K/Protease Digestion #Bisulfite Conversion #Heat Denature #MDA #Ethanol Precipitation #Nextera Library Construction (Low input protocol) #PAGE Analysis ==Protocol== ==Proteinase K/Protease Digestion== *proteinase k *cells *200 5million cells 20ul proteinase k 20mg/ml 400mg *Protease 20mg/ul 56C *QIAGEN Protease (5AU/ml=5mAU/ul), use 0.5mAU for protein digestion *Prepare 1:10 Protease(0.5mAU/ul) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''A''' | align="center" style="background:#f0f0f0;"|'''B''' | align="center" style="background:#f0f0f0;"|'''C''' | align="center" style="background:#f0f0f0;"|'''D''' | align="center" style="background:#f0f0f0;"|'''E''' | align="center" style="background:#f0f0f0;"|'''F''' |- | M-Digestion Buffer (2X)||10||10||10||10||10||10 |- | Sample||1.2||1.2||1.2||1.2||0||0 |- | 1:10 Proteinase K||1||0||0||0||0||0 |- | Proteinase K||0||1||0||0||1||0 |- | 0.5mAU/ul Protease||0||0||0||1||0||0 |- | UV-RT PCR Grade Water||7.8||7.8||8.8||7.8||9||10 |- | Total||20||20||20||20||20||20 |} *Incubate all samples at 50C for 20min *incubate tube D at 70C for 5min for inactivation ==Bisulfite Conversion== *Add 130ul CT Conversion Reagent to all samples *Perform reaction in thermocycler #Step1 98C, 8m #Step2 64C, 3.5hr #Step3 4C, storage for up to 20 hr *Proceed with Zymo column based method with improvement tweak protocol *Elute with 11ul EB Buffer *Transfer bisulfite converted samples to 0.2ml tubes for MDA *Tube D has higher volume after elution, probably due to extra wash buffer loaded into tube ==Heat Denature== *92C for 30 sec *Transfer to ice immediately ==MDA== *Prepare MDA Master Mix {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume Per Reaction''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | 10X RepliPhi Buffer||2||13.2 |- | 1mM N9 Primer||1||6.6 |- | 25mM dNTP||0.8||5.28 |- | RepliPhi Phi29 (100U/ul)||1||6.6 |- | Klenow Exo Minus (10U/ul)||0.5||3.3 |- | H2O||4.7||31.02 |- | Total||10||66 |} *Add 10ul of MDA mastermix to each sample {| {{table}} | align="center" style="background:#f0f0f0;"|'''Plate Layout''' | align="center" style="background:#f0f0f0;"|'''Plate Layout''' |- | ||1 |- | A||A |- | B||B |- | C||C |- | D||D |- | E||E |- | F||F |- | G||Empty |- | H||Empty |- | Plate Layout||Plate Layout |} 30C 60 min, 85C 3 min, 4C hold ==Ethanol Precipitation == *Add 2.5X 100% Ethanol *Add 0.1X 3M NaOAC *Add 1ul glycoblue *Store at -80C for 30min (CONTINUE ON 7/5) *Spin down at 10,000rpm at 4C for 15min *Remove all liquid *Add 500ul 4C chilled 75% ethanol *Spin at 10,000rpm at 4C for 5 min *Remove all liquid *Let dry in hood for 5 min *Add 8ul of nuclease free H2O (aliquot 2ul to perform PCR Analysis) *Let sit at RT to dissolve the pellet *Sucked pellet through the vacuum, only a small piece left ==Nextera Library Construction (Low input protocol)== *In a qPCR tube, mix *3ul genomic DNA *1ul 1:5 Nextera illumina compatible transposomes (10 μl Nextera enzyme, 20 μl 1XTE, 20 μl 100% glycerol) *1ul 5X NExtera HMW buffer *Incubate at 55C for 5 min *Setup PCR Reaction with Klenow Exo- but without primers {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume Per Reaction''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | Water||17.5||115.5 |- | DNA||5||Do not add to MM |- | KAPA Supermix||25||165 |- | Klenow Exo Minus||0.5||3.3 |- | 10uM Orange Primer||1||Do not add to MM |- | 10uM Blue Primer||1||Do not add to MM |- | Total||50||283.8 |} *Add 43ul MM to each reaction *Incubate reactions at RT for 5 min *Put the tubes back on ice *Add Primers *Perform thermal cycling Perform Kapa PCR Reaction 95C 3m 95C 10s 58C 30s 72C 3m plate read Goto step 2 repeat 34 72C 10m Hold 15C ===PAGE Analysis=== [[File:ZhangLab_2011-07-06_1.jpg]] [[File:ZhangLab_2 2011-07-06 10hr 26min.jpg|300px]] ===Results=== *All of the samples were amplified *D had quite a bit of sample lost due to the error during bisulfite column purification *When comparing E & F, it appears that there is a contamination problem with the proteinase K, F has less product compared to E *When comparing A & B, it appears that using 0.1ulproteinase K yields more end product #A-600pg gDNA W 0.1ul proteinase K #B-600pg gDNA W proteinase k #C-600pg gDNA W/O proteinase k #D-600pg gDNA W protease #E-NTC W proteinase K #F-NTC W/O proteinase K ==Bisulfite efficiency PCR Analysis== *Mix 3 MM for CHR8, 21 and 22 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume per Reaction''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | 2X iQ Super Mix||20||132 |- | 3.3uM Primer F ||6||39.6 |- | 3.3uM Primer R||6||39.6 |- | gDNA||2||Do not add to MM |- | H2O||6||39.6 |- | Total||40||264 |} Perform PCR reaction in thermocycler #96C, 3m #95C, 30s #62C, 1m #72C, 1m #Go to step2 repeat 39 times #72C, 5m #4C, Forever ===Page Analysis=== [[File:ZhangLab_2 2011-07-06 15hr 57min.jpg|400px]] [[File:ZhangLab_2 2011-07-06 16hr 00min.jpg|400px]] #A-600pg gDNA W 0.1ul proteinase K #B-600pg gDNA W proteinase k #C-600pg gDNA W/O proteinase k #D-600pg gDNA W protease #E-NTC W proteinase K #F-NTC W/O proteinase K ===Results=== *A All failed (May be working on CHR21, cannot really tell since the image looks weird) *B CHR22 only *C All worked *D CHR8 & CHR21 only *E CHR21 only *F CHR22 only ==To do== *Incorporate the protease digestion after tagmentation to improve yield *Repeat experiment without proteinase K digestion *Repeat experiment with 100 cells with 600pg gDNA as positive control
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