Chris:LabNotes/sci-Methyl Seq/Calendar/2017/2017-6-12
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sci-Methyl Seq Barcode 1 Design v3
Background
- Based on previous tests, it looks as though the original plan of second strand synthesis with an annealed ssDNA Adapter 2 doesn't work very well (it creates many unexpected band sizes). Consequently, in order to address this challenge, we want to redesign how we add Adapter 2 to our DNA fragments.
- We propose the following experimental pipeline, which we will be testing in parallel with the original annealing/second strand synthesis protocol:
End Repair/dA-Tailing 5' -----A 3' 3' A----- 5' | V Add Adpt1_v3: 5' /5Phos/-----TTDD[Barcode1]-----CC 3' 3' T-----AADD[Barcode1]----- 5' | V Ligate Adpt1_v3 (using same optimized ligation protocol as before) 5' -----[Barcode1]DDAA-----T|-----A|-----TTDD[Barcode1]-----CC 3' 3' CC-----[Barcode1]DDTT-----|A-----|T-----AADD[Barcode1]----- 5' | V Add Adpt2_v2: 5' /5Phos/-----[Barcode2]DDDDDDDD----- 3' 3' GG-----[Barcode2]DDDDDDDD----- 5' | V Ligate Adpt2_v2 (using same optimized ligation protocol as before) Nick V 5' -----DDDDDDDD[Barcode2]-----GG|-----[Barcode1]DDAA-----T|-----A|-----TTDD[Barcode1]-----|CC-----[Barcode2]DDDDDDDD----- 3' 3' -----DDDDDDDD[Barcode2]-----CC|-----[Barcode1]DDTT-----|A-----|T-----AADD[Barcode1]-----|GG-----[Barcode2]DDDDDDDD----- 5' ^ Nick | V Denature/separate fragments (will break up DNA at nicks) 5' -----[Barcode1]DDAA-----T|-----A|-----TTDD[Barcode1]-----|CC-----[Barcode2]DDDDDDDD----- 3' 3' -----DDDDDDDD[Barcode2]-----CC|-----[Barcode1]DDTT-----|A-----|T-----AADD[Barcode1]----- 5'