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Fig. 1 | Genome Medicine

Fig. 1

From: Simple paired heavy- and light-chain antibody repertoire sequencing using endoplasmic reticulum microsomes

Fig. 1

Overview of paired antibody HC-LC amplification using microsomes in water-in-oil emulsion droplets. a Antibody-expressing cell populations were used for microsome preparation. b Cells were lysed using a sucrose buffer with 5% digitonin and microsomes with rER-associated mRNAs were enriched using differential centrifugation. c Transmission electron microscopy showed enriched rER microsomes with multilamellar and unilamellar structures. The image was acquired from HEK 293T microsomes used for establishment of the method. Scale bar represents 100 nm. d HC and LC mRNAs were assembled by overlap extension RT-PCR to generate natively paired HC-LC amplicons using constant region primers for reverse transcription and variable region primers for overlap extension assembly. The location and orientation of the paired-end MiSeq reads on the amplicons are indicated by red arrows. e The assembly reaction was carried out within individual emulsion droplets with microsomes from single cells for clonal assembly of rER-associated mRNAs. f Nested PCR amplification with hybridoma-specific nested primers on the assembled DNA demonstrated strong enrichment of native HC-LC pairs when using emulsion PCR during the assembly reaction (upper panel), while a control showed random pairing of heavy and light chains when using conventional open PCR during the assembly reaction

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