Table 1. Oligonucleotides used for RCA and in in situ PLA.
| Oligonucleotide | Description | Vendor | DNA sequence |
|---|---|---|---|
| A | Detection oligo Texas Red | Integrated DNA Technology | 5' – CAGTGAATGCGAGTCCGTCTZZZZ – 3' |
| B | Compaction oligo | Integrated DNA Technology | 5' – AGAGAGTAGTACAGCAGCCGTAAAAGAGAGTAGTACAGCAGCCGTZZZb – 3' |
| Ca | Long circularization oligo | Integrated DNA Technology | 5' – CTATTAGCGTCCAGTGAATGCGAGTCCGTCTA AGAGAGTAGTACAGCAGCCGTCAAGAGTGTCTA – 3' |
| Da | Short circularization oligo | Integrated DNA Technology | 5' – GTTCTGTCATATTTAAGCGTCTTAA – 3' |
| E | Biotinylated RCA template | Eurogentech | 5' – biotin-AAAAAAAAAATATGACAGAACTAGACACTCTT – 3' |
| Fa | Long circularization oligo for Cy3 | Integrated DNA Technology | 5' – CTATTAGCGTCAAGAGAGTAGTACAGCAGCCGTATCAGTG AATGCGAGTCCGTCTAACTAGTGCTGGATGATCGTCCAAGAGT GTCTA – 3' |
| Ga | Long circularization oligo for FITC and Cy5 | Integrated DNA Technology | 5' – CTATTAGCGTCAAGAGAGTAGTACAGCAGCCGTATCAGTG AATGCGAGTCCGTCTAAAGCGATCTGCGAGACCGTATAAGAGT GTCTA – 3' |
| H | Ligation template | Biomers | 5' – GACGCTAATAGTTAAGACGCTTZZZb – 3' |
| I | Detection oligo Cy3 | Integrated DNA Technology | 5' – Cy3-CTAGTGCTGGATGATCGTCCZZZZb – 3' |
| J | Detection oligo FITC | Integrated DNA Technology | 5' – FITC-AGCGATCTGCGAGACCGTATZZZZb– 3' |
| Ka | Padlock probe | Integrated DNA Technology | 5' – GTTCTGTCATACAGTGAATGCGAGTCCGTCTAA GAGAGTAGTACAGCAGCCGTCAAGAGTGTCTA – 3' |
| L | Detection oligo Alexa 488 | Integrated DNA Technology | 5' – Alexa488-AAAAAACAGTGAATGCGAGTCCGTCTZZZZb – 3' |
| M | Ligation template | Integrated DNA Technology | 5' – biotin-CTCTCTCTCTCTCTCTCTCTGTTCACGCTCACCGT GCCCAGTGAGCGAGGACTGCAGCGTAGACG – 3' |
| Na | Padlock probe | Integrated DNA Technology | 5' – CACTGGGCACGGTGAGTGTATGCAGCTCCTC AGTAATAGTGTCTTACAAATCAGTCATACGAGCGCCGCTGCA GTCCTCGCT – 3' |
| O | Ligation template | Integrated DNA Technology | 5' – GACGCTAATAGTAGACACTCTT – 3' |
| P | Detection oligo Cy5 | Integrated DNA Technology | 5' – Cy5-AGCGATCTGCGAGACCGTATZZZZb – 3' |
| Q | Detection oligo Alexa 642 | Integrated DNA Technology | 5'– Alexa642-AGCGATCTGCGAGACCGTATZZZb – 3´ |
aThe oligonucleotide was phosphorylated at a concentration of 2.5 mM prior to use in a buffer containing 1 mM ATP (Thermo Scientific), 1x Reaction buffer A (Thermo Scientific) and 1 U/μl T4 Polynucleotide Kinase (EK0031; Thermo Scientific) for 30 min at 37 °C. The kinase was then heat inactivated at 65 °C for 15 min.
bZ represents 2’O-methyl-RNA Uracil.