Hosuk:FISSEQ Primer information

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Revision as of 19:12, 13 February 2014 by >Mzcai (→‎Detection Probes)
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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/


  • MALAT1_1stRolonyFISH : Cy3 labeled probe for MALAT1 on 1st Rolony
    • /5Cy3/AATGGAGGTATGACATATAATCT


  • 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


  • dcProbe3-Cy3 : for 2nd Rolony
    • Cy3-CCGTTTGATCGTTCGACCGAGA


  • dcProbe4-Cy3 : for 2nd Rolony
    • Cy3-CTACCATTCGCGTGCGATTCCA


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


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


  • dcProbe2RevComp-Cy3 : ATTO488 labeled probe for detecting the reverse complementary sequence of dcProbe2 used for detecting ppMALAT1 and any other padlock probe before RCA with dcProbe2 in barcode
    • TGGTCTGACGCGACGAAGTAGA/3ATTO488N/