scFv-Fc lentiviral library: amplicone 1 (IGH)
The lysed sorted Jurkat cells containing enriched scFv libraries were used to prepare enriched VH-VL amplicons. To avoid skewed distribution of the enriched VH-VL sequences and perform efficient DNA amplification, we utilized emulsion PCR for DNA amplification as previously described with primers, complementary to pLV2-Fc-MTA vector backbone. The obtained VH-VL amplicon was used in two separate PCR reactions to generate overlapping amplicons covering heavy and light IG chains. The overlap contains IG heavy chain CDR3 used for chain pairing after sequencing. Each 25 l PCR reaction contained 5 ng DNA, 200 M of each of dNTP, 1x Tersus polymerase in 1x Tersus Buffer (Evrogen, Russia), and 0.2 M of either Len-pLV2 Amp1 For (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGTCTTGCACTTGTCACGAAT)/Len-pLV2 Amp1 Rev (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAACCGCCTCCACCTAAGC) primers to generate amplicon I or Len-pLV2 Amp2 For (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACAAGATTTGGGCTCGCTA) primers combined with IGH multiplex primer mix (MiLaboratories, USA) to generate amplicon II. The amplification profile was: 2 minutes at 95C followed by 12 cycles of 20s at 95 C, 20s at 55 C, 40s at 72 C and final extension for 2 minutes at 72C. One l of the obtained PCR product was used in the second 25 l PCR reaction containing 200 M of each dNTP, 1 l of Unique Dual Indexing primer mix (Illumina) and 1x Tersus polymerase in 1x Tersus Buffer amplified for 6 cycles using the same amplification profile as for the first PCR. The obtained libraries were pooled and sequenced on Illumina Miseq (paired end 300+300).