Table 1.
List of oligonucleotides used for PAT preparation | ||
---|---|---|
Designation | Sequence (5’ -> 3’) | Use |
Primers used for Method 1 | ||
RT-PE1 series | 5’-biotin–TCCTGCTGAACCGCTCTTCCGATCTNNNXXTTTTTTTTTTTTTTTTTTVN | Reverse transcription primer |
RT-PE2 series | TCCTGCTGAACCGCTCTTCCGATCTNNNXXTTTTTTTTTTTTTTTTTTVN | Reverse transcription primer |
PE-RTbio | 5’-biotin TCCTGCTGAACCGCTCTTCCGATCT | Second strand synthesis |
SWITCH1.1 | Strand switching | |
SWITCH1.2 | Second strand synthesis | |
PE-ADN1A | Ligation of 5’ Illumina adapter | |
PE-ADT1A | Ligation of 5’ Illumina adapter | |
PE-AD2A | AGATCGGAAGAGCGTCGTGTAGTCTGTCGAGAC | Ligation of 5’ Illumina adapter |
PE-ADN1B | Ligation of 5’ Illumina adapter | |
PE-ADT1B | Ligation of 5’ Illumina adapter | |
PE-AD2B | TCAGATCGGAAGAGCGTCGTGTAGTCTGTCGAGAC | Ligation of 5’ Illumina adapter |
PE-ADN1C | Ligation of 5’ Illumina adapter | |
PE-ADT1C | Ligation of 5’ Illumina adapter | |
PE-AD2C | AGAGATCGGAAGAGCGTCGTGTAGTCTGTCGAGAC | Ligation of 5’ Illumina adapter |
PE-ADN1D | Ligation of 5’ Illumina adapter | |
PE-ADT1D | Ligation of 5’ Illumina adapter | |
PE-AD2D | GGAGATCGGAAGAGCGTCGTGTAGTCTGTCGAGAC | Ligation of 5’ Illumina adapter |
Primers used for Methods 2A and 2B | ||
RT-PE3 series | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNXXXTTTTTTTTTTTTTTTTTTVN | Reverse transcription primer |
SWITCH2 | CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGGG | Strand switching |
Primers used to amplify tags | ||
PE-PCR1 | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT | Tag amplification |
PE-PCR2 | CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT | Tag amplification |
“N” – random base
“V” – A, C, or G, randomized in the primer population
“XX”, “XXX” – positions of bar codes used to distinguish samples for multiplex sequencing.