Hosuk:FISSEQ Primer information

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Primers[edit]

  • 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[edit]

Structure[edit]

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


Padlock probes[edit]

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


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


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


  • ppMALAT1_NH2
    • /5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGG/iUniAmM/TCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT


  • MALAT1_template
    • /5AmMC6/GAATTGATAAGTAAAGGCAGAAAAGATTATATGTCATACCTCCAT
  • ppCOL1A1 (dcProbe3-Cy3)
    • /5Phos/CCTCAGCCTCCTGACTAGCTGCTTCAGCTTCCCGATATCCGACGGCCGTTTGATCGTTCGACCGAGACCGGGTTCATGCAATTCTCCTG

Detection Probes[edit]

  • 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/

Agi26k Probeset Barcode-to-dcProbe Key[edit]

[Position][Color] Sequence dcProbe
[0][0]
[0][1] ATGTATCGCGCTCGATTGGCAG dcProbe0-FAM
[0][2] ACGTATCGGTAGTCGCAACGCA dcProbe0-Cy3
[0][3] TACGCTACGGAGTACGCCACTC
[1][0]
[1][1] GTCTTGCGTGCGATACGGAGTA dcProbe1-Cy3
[1][2] CAACGGTATTCGGTCGTCATCC dcProbe1-FAM
[1][3] ACTGGTTCGGGCGTACCTAACA
[2][0]
[2][1] TAGAACTTGCGCGGATACACGG dcProbe2-FAM
[2][2] TCTACTTCGTCGCGTCAGACCA dcProbe2-Cy3
[2][3] GGACGAACGGTCGAGATTTACC
[3][0]
[3][1] CGAATTGTCCGCGCTCTACGAT dcProbe3-FAM
[3][2] CCGTTTGATCGTTCGACCGAGA dcProbe3-Cy3
[3][3] AAACTGCGACCGTCGGCTTACT
[4][0]
[4][1] TCGGAATACGTCGTTGACTGCG dcProbe4-FAM
[4][2] CTACCATTCGCGTGCGATTCCA dcProbe4-Cy3
[4][3] CCAGGGATCGGTCGAGTACGCT
[5][0]
[5][1] TGAGTGTCGCGCAACTTAGCGG
[5][2] AACGTCTGCGTACCGGCTTAGC
[5][3] ACATGCGATTAACCGCGACTGC
[6][0]
[6][1] TCACGCTTACGATCCCGCTATG
[6][2] TTCGTAACCCGTGCGAAGTGCC
[6][3] CCTCTCGTAGCGTGCGATGAGA
  • Position [7] (8th position if count first as 1) not used

Agi26k Probeset Barcode-to-dcProbe Key for dcProbes Ordered 5/14/14[edit]

[Position][Color] Sequence dcProbe
[0][0]
[0][1] ATGTATCGCGCTCGATTGGCAG dcProbe0-488
[0][2] ACGTATCGGTAGTCGCAACGCA dcProbe0-Cy3
[0][3] TACGCTACGGAGTACGCCACTC dcProbe0-Cy5
[1][0]
[1][1] GTCTTGCGTGCGATACGGAGTA dcProbe1-488
[1][2] CAACGGTATTCGGTCGTCATCC dcProbe1-Cy3
[1][3] ACTGGTTCGGGCGTACCTAACA dcProbe1-Cy5
[2][0]
[2][1] TAGAACTTGCGCGGATACACGG dcProbe2-488
[2][2] TCTACTTCGTCGCGTCAGACCA dcProbe2-Cy3
[2][3] GGACGAACGGTCGAGATTTACC dcProbe2-Cy5
[3][0]
[3][1] CGAATTGTCCGCGCTCTACGAT dcProbe3-488
[3][2] CCGTTTGATCGTTCGACCGAGA dcProbe3-Cy3
[3][3] AAACTGCGACCGTCGGCTTACT dcProbe3-Cy5
[4][0]
[4][1] TCGGAATACGTCGTTGACTGCG dcProbe4-488
[4][2] CTACCATTCGCGTGCGATTCCA dcProbe4-Cy3
[4][3] CCAGGGATCGGTCGAGTACGCT dcProbe4-Cy5
[5][0]
[5][1] TGAGTGTCGCGCAACTTAGCGG dcProbe5-488
[5][2] AACGTCTGCGTACCGGCTTAGC dcProbe5-Cy3
[5][3] ACATGCGATTAACCGCGACTGC dcProbe5-Cy5
[6][0]
[6][1] TCACGCTTACGATCCCGCTATG dcProbe6-488
[6][2] TTCGTAACCCGTGCGAAGTGCC dcProbe6-Cy3
[6][3] CCTCTCGTAGCGTGCGATGAGA dcProbe6-Cy5