Matt:LabNotes/2014-9-19: Difference between revisions

From ZhangLabWiki
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

' 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