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=== 2013_04_18 === * Dr. Zhang suggested Jeff to try CGI protocol by using dUTP, and suggested me to checked with Rui for the concentration of adapter she used for her mouse PGC RNAseq experiment. * Note from Rui: email on April 18, 2013 ** http://genome-tech.ucsd.edu/LabNotes/index.php/Kun:LabNotes/SingleCellExpr/2011-7-5#PCR_products ** http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Collaborations/2011-7-11#End repair ** http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Collaborations/2011-7-11#Pre-run ** http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Collaborations/2011-7-12#Prepare_RNAseq_library_for_sequencing ** Summary of adapter she used (note: her fragment size ~200bp, mine is 400bp) *** For template <10ng: added 0.08ul of 30uM adapters --> final conc. in 50ul = 0.048uM. This means that if I used 20uM adapters, I should add (50ul*0.048uM)/50ul = 0.12ul *** For template 10ng - 50ng: added 0.4ul of 30uM adapters --> final conc. in 50ul = 0.24uM (5X more than template <10ng) ** Perform qPCR in 50ul reaction, and added 1ul of 10uM each primer. * I have some discussions with Jeff, and he has shared this paper for CGI protocol as Dr. Zhang suggested [http://www.nature.com/nature/journal/v487/n7406/full/nature11236.html]. * I am planning to repeat experiment using low DNA input (1ng, 2ng and 5ng input) and adapter concentration folowing Rui's experiment. I still have sheared gDNA of NA12878 (may need to re-quantify with Qubit assay again to confirm conc.) * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-4-24
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