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]
- The number 1 padlock probe is the same one we ordered from IDT (ppCOL1A1) and is likely to be responsible for 50% of 2nd rolonies seen from Agi26k0gap in situ
- Table is gDNA sorted of Agi26k_Capture_Analysis.xlsx from Matt:LabNotes/2013-8-21
' | 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 |