Hosuk:FISSEQ Primer information: Difference between revisions
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>Hosuki78 No edit summary |
>Mzcai m (→Padlock probes) |
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(10 intermediate revisions by 2 users not shown) | |||
Line 11: | Line 11: | ||
**GATATCGGGAAGCTGA*A*G | **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 | ====Padlock probes==== | ||
*ppACTB | *ppACTB | ||
**H1: | |||
**H2: | |||
**/5Phos/CTGTGCTCGCGGGGCGCTTCAGCTTCCCGATATCCGACGGACGTATCGGTAGTCGCAACGCAGGCAAAGGCGAGGCT | **/5Phos/CTGTGCTCGCGGGGCGCTTCAGCTTCCCGATATCCGACGGACGTATCGGTAGTCGCAACGCAGGCAAAGGCGAGGCT | ||
*ppRAB7A | *ppRAB7A | ||
**H1: | |||
**H2: | |||
**/5Phos/GAAGCGAGAAGGTCCAAGTTCTGCTTCAGCTTCCCGATATCCGACGGGTCTTGCGTGCGATACGGAGTAGAGGAGACTAAACGGAGGACA | **/5Phos/GAAGCGAGAAGGTCCAAGTTCTGCTTCAGCTTCCCGATATCCGACGGGTCTTGCGTGCGATACGGAGTAGAGGAGACTAAACGGAGGACA | ||
*ppMALAT1 | *ppMALAT1 | ||
**/5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGGTCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT | **H1: TTTCTGCCTTTACTTATCAATTC | ||
**H2: AATGGAGGTATGACATATAATCT | |||
**/5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGGTCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT | |||
*ppMALAT1_NH2 | |||
**/5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGG/iUniAmM/TCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT | |||
*MALAT1_template | |||
**/5AmMC6/GAATTGATAAGTAAAGGCAGAAAAGATTATATGTCATACCTCCAT | |||
*ppCOL1A1 (dcProbe3-Cy3) | |||
**/5Phos/CCTCAGCCTCCTGACTAGCTGCTTCAGCTTCCCGATATCCGACGGCCGTTTGATCGTTCGACCGAGACCGGGTTCATGCAATTCTCCTG | |||
===Detection Probes=== | ===Detection Probes=== | ||
Line 42: | Line 64: | ||
*RAB7A_1stRolonyFISH : ATTO550 labeled probe for RAB7A on 1st Rolony | *RAB7A_1stRolonyFISH : ATTO550 labeled probe for RAB7A on 1st Rolony | ||
**GAGGAGACTAAACGGAGGACA/3ATTO550N/ | **GAGGAGACTAAACGGAGGACA/3ATTO550N/ | ||
*MALAT1_1stRolonyFISH : Cy3 labeled probe for MALAT1 on 1st Rolony | |||
**/5Cy3/AATGGAGGTATGACATATAATCT | |||
Line 56: | Line 82: | ||
*dcProbe2-Cy3 : for 2nd Rolony of | *dcProbe2-Cy3 : for 2nd Rolony of ppMALAT1 | ||
**Cy3-TCTACTTCGTCGCGTCAGACCA | **Cy3-TCTACTTCGTCGCGTCAGACCA | ||
*dcProbe3-Cy3 : for 2nd Rolony | |||
**Cy3-CCGTTTGATCGTTCGACCGAGA | |||
*dcProbe4-Cy3 : for 2nd Rolony | |||
**Cy3-CTACCATTCGCGTGCGATTCCA | |||
Line 67: | Line 100: | ||
*RT-Adapt : Cy5 labeled probe for cDNA after RT | *RT-Adapt : Cy5 labeled probe for cDNA after RT | ||
**/5Cy5/TCTTCAGCGTTCCCTAGA | **/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=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''[Position][Color]''' | |||
| align="center" style="background:#f0f0f0;"|'''Sequence''' | |||
| align="center" style="background:#f0f0f0;"|'''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=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''[Position][Color]''' | |||
| align="center" style="background:#f0f0f0;"|'''Sequence''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|} |
Latest revision as of 01:08, 3 September 2014
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 |