Table 2.
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 |
Illumina Nextera index adapters (i5 and i7). See illumina Nextera DNA library preparation reference guide and illumina adapters sequences list for more information.