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. 2021 Feb 25;12:629059. doi: 10.3389/fimmu.2021.629059

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Copyright © 2021 Johansson, Yohannes, Koskela, Partanen and Saavalainen

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Experimental design of Illumina HLA RNA-Seq. In the library preparation process, mRNA is first transcribed into complementary DNA (cDNA) with simultaneous integration of 10 bp unique molecular identifier (UMI) in RNA template switching oligo (rnaTSO) and further amplified. The full-length cDNA (FLcDNA) is then divided and processed in parallel in Illumina’s cDNA and amplicon library preparation protocols. The amplicon library preparation included an enrichment of HLA genes using gene-specific primers. Lastly, the full-length cDNA and HLA amplicons are tagmented, and sample-specific barcodes are added for multiplexing. The method results 5’ end library molecules suitable for Illumina paired-end sequencing.