Table 2.
Oligonucleotides used.
| Primer | Application | Sequence |
|---|---|---|
| FIRST-STRAND cDNA SYNTHESIS | ||
| SmartNNNext | 5′–template-switch oligo with sequencing illumina adapter U = dU, rG = riboG | AGATGUGTAUAAGAGACAGNNNNUNNNNUNNNNUCTT(rG)4 |
| IGH cDNA synthesis primer mix | ||
| hIGG_r1 | Primer for cDNA synthesis, human IgG heavy-chain mRNA | GAAGTAGTCCTTGACCAGGCA |
| hIGM_r1 | Primer for cDNA synthesis, human IgM heavy-chain mRNA | GTGATGGAGTCGGGAAGGAAG |
| hIGA_r1 | Primer for cDNA synthesis, human IgA heavy-chain mRNA | GCGACGACCACGTTCCCATCT |
| hIGD_r1 | Primer for cDNA synthesis, human IgD heavy-chain mRNA | GGACCACAGGGCTGTTATC |
| hIGE_r1 | Primer for cDNA synthesis, human IgE heavy-chain mRNA | AGTCACGGAGGTGGCATTG |
| FIRST PCR AMPLIFICATION | ||
| Common primer | Step-out primer, anneals on the switch adaptor | AGATGTGTATAAGAGACAG |
| IGH reverse primer mix | ||
| Common-hIGGE_r2 | Nested primer with sequencing illumina adaptor, human IgG/IgE heavy-chain cDNA | AGATGTGTATAAGAGACAGARGGGGAAGACSGATG |
| Common-hIGA_r2 | Nested primer with sequencing illumina adaptor, human IgA heavy-chain cDNA | AGATGTGTATAAGAGACAGCAGCGGGAAGACCTTG |
| Common-hIGM_r2 | Nested primer with sequencing illumina adaptor, human IgM heavy-chain cDNA | AGATGTGTATAAGAGACAGAGGGGGAAAAGGGTTG |
| Common-hIGD_r2 | Nested primer with sequencing illumina adaptor, human IgD heavy-chain cDNA | AGATGTGTATAAGAGACAGATATGATGGGGAACAC |
| SECOND PCR AMPLIFICATION | ||
| F-common | Step-out primer with sequencing and P7 illumina adapters | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG |
| R- common | Step-out primer with sequencing and P5 illumina adapters | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG |
| THIRD PCR AMPLIFICATION | ||
| Fc_i7a | Step-out primer with index 1 illumina adapter | CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG |
| Fc_i5b | Step-out primer with index 2 illumina adapter | AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC |
a, b
Illumina Nextera index adapters (i5 and i7). See illumina Nextera DNA library preparation reference guide and illumina adapters sequences list for more information.