Matt:LabNotes/2014-9-19: Difference between revisions
Jump to navigation
Jump to search
>Mzcai (Created page with "==Suppressing ppCOL1A1 and other COL1A1 probes in Agi26k0gap== *Use suppressor oligos to reduce "high efficiency" padlock probes **Reference: Deng, J. "Targeted bisulfite seq...") |
>Mzcai mNo edit summary |
||
Line 6: | Line 6: | ||
*Oligos are reverse complimentary to extension arm of padlock probe and will out-compete | *Oligos are reverse complimentary to extension arm of padlock probe and will out-compete | ||
**Make Tm 5 degrees higher than reaction temp | **Make Tm 5 degrees higher than reaction temp | ||
===15 Most Efficient Padlock Probes=== | |||
*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 [[Media:Agi26k_Capture_Analysis.xlsx | Agi26k_Capture_Analysis.xlsx]] from [[Matt:LabNotes/2013-8-21]] | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''gDNA counts''' | |||
| align="center" style="background:#f0f0f0;"|'''cDNA counts''' | |||
| align="center" style="background:#f0f0f0;"|'''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=== | |||
*Chose Top 6 because these were abundant in PGP1F Full Decoding with Agi26k0gap | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''gDNA counts''' | |||
| align="center" style="background:#f0f0f0;"|'''cDNA counts''' | |||
| align="center" style="background:#f0f0f0;"|'''Gene''' | |||
| align="center" style="background:#f0f0f0;"|'''Sequence''' | |||
| align="center" style="background:#f0f0f0;"|'''H2 Arm (3' end)''' | |||
| align="center" style="background:#f0f0f0;"|'''Tm of H2''' | |||
| align="center" style="background:#f0f0f0;"|'''Suppressor''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|} |
Revision as of 07:22, 23 September 2014
Suppressing ppCOL1A1 and other COL1A1 probes in Agi26k0gap
- 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
- 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
- Chose Top 6 because these were abundant in PGP1F Full Decoding with Agi26k0gap
' | 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 |