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The 3G vector primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5′ end of RNA transcript) approach.
We hybridized the biotinylated probes to the denatured single-stranded (ss) cDNAs generated by the SMART (switching mechanism at 5' end of RNA transcript) PCR, and thereby isolated the expressed MHC sequences.
The PCR-based cDNA library was made following the instructions for the SMART (switching mechanism at 5' end of RNA transcript) cDNA library construction kit (Clontech).
aa: amino acid; AMP: antimicrobial peptide; AG5: antigen 5 family; EST: expressed sequence tag; H class: housekeeping; NR: nonredundant; OBP: odorant-binding protein; S class: secreted; SG: salivary gland; SMART: switching mechanism at 5' end of RNA transcript; U class: unknown function.
Codon volatility was calculated as previously described [ 62]. aa: amino acid; AMP: antimicrobial peptide; AG5: antigen 5 family; EST: expressed sequence tag; H class: housekeeping; kbase: kilobase; NR: nonredundant; nt: nucleotide; OBP: odorant binding protein; S class: secreted; SG: salivary gland; SMART: switching mechanism at 5/end of RNA transcript; U class: unknown function.
This method, successfully used for eukaryotic single cell transcriptomic studies44,45, converts poly(A)+ RNA to full-length cDNA using a modified oligo dT) primer combined with SMART (Switching Mechanism at the 5′ end of RNA Template) technology.
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