Matt:LabNotes/2014-9-19

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Suppressing ppCOL1A1 and other COL1A1 probes in Agi26k0gap[edit]

  • Use suppressor oligos to reduce "high efficiency" padlock probes
    • Reference: Deng, J. "Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming"
    • Since these are 0gap probes can't use the "bridge to nowhere" strategy
  • Oligos are reverse complimentary to extension arm of padlock probe and will out-compete
    • Make Tm 5 degrees higher than reaction temp

15 Most Efficient Padlock Probes[edit]

' gDNA counts cDNA counts Gene
Probe:12447 571984 23444 COL1A1
Probe:12503 116827 1635 RUNX1
Probe:12226 64099 2742 GEMIN2
Probe:12900 52653 69 HAT1
Probe:12876 16861 50 ERBB3
Probe:12659 15451 160 NUMB
Probe:8986 14267 533 PSG4
Probe:1173 13875 622 CCDC144NL
Probe:8184 12940 56 OR7D4
Probe:8987 11982 685 PSG4
Probe:8995 11912 340 PSG8
Probe:10542 11324 798 SSX1
Probe:8863 8781 66 PRB2
Probe:11662 8193 1286 UGT2B11
Probe:11675 7778 859 UGT2B7

Designed Suppressor Oligos for Top 6[edit]

  • Chose Top 6 because these were abundant in PGP1F Full Decoding with Agi26k0gap
  • Named oligos: supp_<genename> (e.g. supp_COL1A1)
' gDNA counts cDNA counts Gene Sequence H2 Arm (3' end) Tm of H2 Suppressor Tm of Suppressor
Probe:12447 571984 23444 COL1A1 CCTCAGCCTCCTGACTAGCTGCTTCAGCTTCCCGATATCCGACGGTAGTGTTACGCTACGGAGTACGCCACTCTAGAACTTGCGCGGATACACGGAAACTGCGACCGTCGGCTTACTTCGGAATACGTCGTTGACTGCGACATGCGATTAACCGCGACTGCAAAAAAACCGGGTTCATGCAATTCTCCTG CCGGGTTCATGCAATTCTCCTG 64.5 CAGGAGAATTGCATGAACCCGG 64.5
Probe:12503 116827 1635 RUNX1 GCAATTCTCCTGTCTTGGCCTCCTTCAGCTTCCCGATATCCGACGGTAGTGTTACGCTACGGAGTACGCCACTCTCTACTTCGTCGCGTCAGACCACGAATTGTCCGCGCTCTACGATCTACCATTCGCGTGCGATTCCACCTCTCGTAGCGTGCGATGAGAAAAACTGGTCTCAATCTCCTGGACTCAA CTGGTCTCAATCTCCTGGACTCAA 64.13 TTGAGTCCAGGAGATTGAGACCAG 64.13
Probe:12226 64099 2742 GEMIN2 GCTGAGGTGGGAGGATTGCTGCTTCAGCTTCCCGATATCCGACGGTAGTGTTACGCTACGGAGTACGCCACTCTCTACTTCGTCGCGTCAGACCATCGGAATACGTCGTTGACTGCGACATGCGATTAACCGCGACTGCTCACGCTTACGATCCCGCTATGAAAAAGTTGTGGTCCTAGCTACTCAGGAG GTTGTGGTCCTAGCTACTCAGGAG 64.43 CTCCTGAGTAGCTAGGACCACAAC 64.43
Probe:12900 52653 69 HAT1 TTTAGTAGAGACAGGGTTTCACTTCAGCTTCCCGATATCCGACGGTAGTGTTACGCTACGGAGTACGCCACTCTCTACTTCGTCGCGTCAGACCACTACCATTCGCGTGCGATTCCATGAGTGTCGCGCAACTTAGCGGTTCGTAACCCGTGCGAAGTGCCAAAAAAAAAGCCTAGCTAATGTTTGTATT AAGCCTAGCTAATGTTTGTATT 56.12 AATACAAACATTAGCTAGGCTTTTTTTTTGGC 64.67
Probe:12876 16861 50 ERBB3 AGAGAGAGGGAGGGAGGAACGCTTCAGCTTCCCGATATCCGACGGTAGTGTTACGCTACGGAGTACGCCACTCTAGAACTTGCGCGGATACACGGAAACTGCGACCGTCGGCTTACTCCAGGGATCGGTCGAGTACGCTACATGCGATTAACCGCGACTGCAAAAAAAAATGTGTGTGTGTGAGAGAGAG TGTGTGTGTGTGAGAGAGAG 59.67 CTCTCTCTCACACACACACATTTTTTTTTG 64.32
Probe:12659 15451 160 NUMB CTCCTGGCCTCAAGTGATGTTCTTCAGCTTCCCGATATCCGACGGTAGTGTTACGCTACGGAGTACGCCACTCTCTACTTCGTCGCGTCAGACCACGAATTGTCCGCGCTCTACGATTCGGAATACGTCGTTGACTGCGTCACGCTTACGATCCCGCTATGAAAACTATGTTGCACAGTCTGGTCACAAA CTATGTTGCACAGTCTGGTCACAAA 64.12 TTTGTGACCAGACTGTGCAACATAG 64.12