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=Priorities= 1) Nature method paper * (<6days) rebuttal - google doc - address issues - disclose and highlight probes design * demonstrate that probes will work on LC sciences oligos - $1100, 4K, 100 mer will take 2 weeks - (<6days)Target selections: epiMemory, epiMutation sets, +immunogenicity genes - Immunogenicity - Yang Xu paper: '''Hormad1, Zg16''' A. Hormad1 - chr1:148,937,166-148,959,976 (~22kbp) DNA sequence [http://genome.ucsc.edu/cgi-bin/hgc?hgsid=210264721&g=htcGetDna2&table=&i=mixed&o=148937165&l=148937165&r=148959976&getDnaPos=chr1%3A148%2C937%2C166-148%2C959%2C976&db=hg18&hgSeq.cdsExon=1&hgSeq.padding5=0&hgSeq.padding3=0&hgSeq.casing=upper&boolshad.hgSeq.maskRepeats=0&hgSeq.repMasking=lower&boolshad.hgSeq.revComp=0&submit=get+DNA] B. Zg16 - chr16:29,697,062-29,700,470 (~3kbp) DNA sequence [http://genome.ucsc.edu/cgi-bin/hgc?hgsid=210264721&g=htcGetDna2&table=&i=mixed&o=29697061&l=29697061&r=29700470&getDnaPos=chr16%3A29%2C697%2C062-29%2C700%2C470&db=hg18&hgSeq.cdsExon=1&hgSeq.padding5=0&hgSeq.padding3=0&hgSeq.casing=upper&boolshad.hgSeq.maskRepeats=0&hgSeq.repMasking=lower&boolshad.hgSeq.revComp=0&submit=get+DNA] - Cross check with Ecker's cg_dmrs in ALL iPSCs versus ESCs list: [[File:nature097980-s2-cg_dmr_alliPSCs.csv]] [[File:PGP1iPS_memoryXList_highPriority.txt]] - 41 [[File:PGP1iPS_mutationXList_highPriority.txt]] - 10 - Cross check with Ecker's cg_dmrs in any iPSCs versus ESCs list: [[File:nature09798-s2-cg_dmr.csv]] C. [[File:PGP1iPS_memoryXList.txt]] - (include 41 from above) 236 (~16% of memory sites) D. [[File:PGP1iPS_mutationXList.txt]] - (include 10 from above) 101 (~11% of memory sites) - All memory and mutation sites in PGP1iPS [[File:PGP1iPS_memory_methylMatrix.txt]] - 1476 sites [[File:PGP1iPS_mutation_methylMatrix.txt]] - 904 sites - how far does each spread? 1-6.4 kbp (ranges of cg_dmrs in files above) *Uniquely barcoded probes - (<10days)orig probes -> inverted probes + barcode -> bc probes -> prep + sanger validation *Uniquely barcoded circles - (<7-10days) Uracil F primer-> 2 cycles PCR -> control elongation time and temperature * add limited amount of primers to improve ease of digestion. - USER digestion - Add short primer - continue for several cycles - clonal sequencing - 10 clones *2x2 Protocol test Zymo versus Imprint (perform bisulfite conversion using same DNA) Circles amplification with and without USER digestion * Side project: Peak calling pipeline for 5cC enrichment analysis. 2) N37 10 tissues - data analysis 3) UPenn -> PCA, and mQTL (leave running for 2 weeks) 4) Episomal reprogramming 5) Sergio's samples 6) HIV
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