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==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||154 |- | DNA||5||Do not add to MM |- | KAPA Supermix||25||220 |- | Klenow Exo Minus||0.5||4.4 |- | 10uM Orange Primer||1||Do not add to MM |- | 10uM Blue Primer||1||Do not add to MM |- | Total||50||378.4 |} *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 [[File:ZhangLab_2011-06-29_1.JPG|700px]] #A1-100 sperm cells #A2-100 sperm cells (repeat) #B1-100 ESCs #B2-100 ESCs (repeat) #C1-600pg GM12878 gDNA #D-1.2ng GM12878 gDNA #E1-NTC #E2-NTC(MDA)
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