Hosuk:FISSEQ Primer information: Difference between revisions

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*FISSEQ_ppRCA : RCA primer for 2nd Rolony fab. from padlock probes
*FISSEQ_ppRCA : RCA primer for 2nd Rolony fab. from padlock probes
**GATATCGGGAAGCTGA*A*G
**GATATCGGGAAGCTGA*A*G




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**H2: AATGGAGGTATGACATATAATCT  
**H2: AATGGAGGTATGACATATAATCT  
**/5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGGTCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT
**/5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGGTCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT


===Detection Probes===
===Detection Probes===

Revision as of 19:00, 12 November 2013

Primers

  • FISSEQ_RT : RT primer for 1st Rolony fabrication
    • /5Phos/TCTCGGGAACGCTGAAGANNNNNN


  • FISSEQ_RCA : RCA primer for 1st Rolony fabrication
    • TCTTCAGCGTTCCCGA*G*A


  • FISSEQ_ppRCA : RCA primer for 2nd Rolony fab. from padlock probes
    • GATATCGGGAAGCTGA*A*G


Padlock Probes

Structure

  • Probe: H1 + Common seq. for 2nd RCA + Common Linker + decoding probe seq. + H2
  • Common seq. for 2nd RCA : CTTCAGCTTCCCGATATC
  • Common Linker : CGACGG


Padlock probes

  • ppACTB
    • H1:
    • H2:
    • /5Phos/CTGTGCTCGCGGGGCGCTTCAGCTTCCCGATATCCGACGGACGTATCGGTAGTCGCAACGCAGGCAAAGGCGAGGCT


  • ppRAB7A
    • H1:
    • H2:
    • /5Phos/GAAGCGAGAAGGTCCAAGTTCTGCTTCAGCTTCCCGATATCCGACGGGTCTTGCGTGCGATACGGAGTAGAGGAGACTAAACGGAGGACA


  • ppMALAT1
    • H1: TTTCTGCCTTTACTTATCAATTC
    • H2: AATGGAGGTATGACATATAATCT
    • /5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGGTCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT


Detection Probes

  • FISSEQ_Adpt : Cy3 labeled probe for 1st Rolony detection
    • /5Cy3/TCTCGGGAACGCTGAAGA


  • FISSEQ_AdptCy5 : Cy5 labeled probe for 1st Rolony detection
    • /5Cy5/TCTCGGGAACGCTGAAGA


  • ACTB_1stRolonyFISH : ATTO488 labeled probe for ACTB on 1st Rolony
    • GGCAAAGGCGAGGCT/3ATTO488N/


  • RAB7A_1stRolonyFISH : ATTO550 labeled probe for RAB7A on 1st Rolony
    • GAGGAGACTAAACGGAGGACA/3ATTO550N/


  • Shifted FISSEQ_2ndRCAprimerDye : 2nd Rolony from all padlock probes
    • GGATATCGGGAAGCTGAA/3ATTO488N/


  • dcProbe0-Cy3 : for 2nd Rolony of ppACTB
    • Cy3-ACGTATCGGTAGTCGCAACGCA


  • dcProbe1-Cy3 : for 2nd Rolony of ppRAB7A
    • Cy3-GTCTTGCGTGCGATACGGAGTA


  • dcProbe2-Cy3 : for 2nd Rolony of ppMALAT1
    • Cy3-TCTACTTCGTCGCGTCAGACCA


  • RT-dT50 : Cy3 labeled probe for mRNA detection
    • /5Cy3/TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT


  • RT-Adapt : Cy5 labeled probe for cDNA after RT
    • /5Cy5/TCTTCAGCGTTCCCTAGA