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==Overview== *Below is a general overview of the experimental procedure to ligate Adpt1 and Adpt2 (same as <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/sci-Methyl_Seq/Calendar/2017/2017-6-12> but with PCR added in). '''NOTE: <span style="color:red">NoG</span> sequences are in <span style="color:red">red/H</span>, <span style="color:blue">NoC</span> sequences are in <span style="color:blue">blue/D</span>.''' <u>End Repair/dA-Tailing</u> 5' '''-----'''A 3' 3' A'''-----''' 5' | V Add Adpt1_v4: 5' /5Phos/<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span> 3' 3' <u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span> 5' | V <u>Ligate Adpt1_v4 (using same optimized ligation protocol as before)</u> 5' <span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span> 3' 3' <span style="color:red">CC-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span> 5' | V <u>Add Adpt2_v3:</u> 5' /5Phos/<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span> 3' 3' <span style="color:red">GG-----[Barcode2]'''HHHHHHHH'''-----</span> 5' | V <u>Ligate Adpt2_v3 (using same optimized ligation protocol as before)</u> Nick V 5' <span style="color:red">-----'''HHHHHHHH'''[Barcode2]-----GG</span>|<span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span> 3' 3' <span style="color:blue">-----'''''DDDDDDDD'''''[''Barcode2'']-----</span>|<span style="color:red">CC-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span>|<span style="color:red">GG-----[Barcode2]'''HHHHHHHH'''-----</span> 5' ^ Nick | V <u>Denature/separate fragments (will break up DNA at nicks)</u> 5' <span style="color:blue">-----[''Barcode1'']DDAA-----</span><u>ACT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----CC</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span> 3' 3' <span style="color:blue">-----'''''DDDDDDDD'''''[''Barcode2'']-----</span>|<span style="color:red">CC-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TCA</u><span style="color:blue">-----AADD[''Barcode1'']-----</span> 5' | V <u>Bisulfite conversion</u> (C->U) | V <u>PCR (P7 will be added first followed by P5)</u> P5: 5' AATGATACGGCGACCACCGA 3' P7: 5' CAAGCAGAAGACGGCATACGAGAT 3' 5' <span style="color:blue">-----[''Barcode1'']''DD''AA-----</span><u>AUT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----UU</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''----- 3'</span> ---------------------------------------------------------------------------------- 3' <----- -----> 3' \ / P7 5' 5' P5 P5 5' P7 / \ <----- -----> 3' ---------------------------------------------------------------------------------- 3' <span style="color:blue">-----'''''DDDDDDDD'''''[''Barcode2'']-----</span>|<span style="color:red">UU-----[Barcode1]HHTT-----</span><u>TG</u>|A'''-----'''|<u>TUA</u><span style="color:blue">-----AA''DD''[''Barcode1'']----- 5' </span> | V <u>PCR Product</u> 5' P5<span style="color:blue">-----[''Barcode1'']''DD''AA-----</span><u>AAT</u>|'''-----'''A|<u>GT</u><span style="color:red">-----TTHH[Barcode1]-----AA</span>|<span style="color:blue">-----[''Barcode2'']'''''DDDDDDDD'''''-----</span>P7 3' -----> -----> -----> Index2 Primer Read1 Primer Index2 Primer
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