Reverse Transcription by Template Switching and Target Amplification

We used an adaptation of the STRT method to generate full length complementary DNA (cDNA) molecules from RNA transcripts (41). Briefly, the poly-A hybridization to the first strand cDNA synthesis primer was performed in a 96-well plate in a T100™ Thermal Cycler (Biorad) with 3 min at 72°C with 25 ng of RNA, 1% Triton™ X-100 (Sigma), 20 µM of STRT-V3-T30-VN oligo, 100 µM of DTT (invitrogen, life technologies, Thermo Fisher), 10 mM dNTP (Bioline), 4 U of Recombinant RNase Inhibitor (Takara Clontech), 1:1,000 The Ambion^® ERCC RNA Spike-In Control Mix (life technologies, Thermo Fisher) in a total volume of 3 µl. All oligos were from Integrated DNA Technologies and are listed in Table S1 . Reverse transcription of the whole transcriptome was performed adding 3.7 µl of the RT mix containing 5× SuperScript first strand buffer (invitrogen by Thermo Fisher Scientific), 1 M MgCl₂ (Sigma), 5 M Betaine solution (Sigma), 134 U of SuperScript ^® II Reverse Transcriptase (invitrogen by Thermo Fisher Scientific), 40 µM RNA-TSO 10bp UMI, 5.6 U of Recombinant RNase Inhibitor immediately to each reaction. To complete the reverse transcription and the template switching, the plate was incubated 90 min at 42°C followed by 10 min at 72°C. In this reaction, every transcript receives a unique distinct barcode. After RT the cDNA was further amplified with 2× KAPA HiFi HotStart ReadyMix (Kapa Biosystems), 10 µM ImSTRT-TSO-PCR with a thermal profile consisted of an initial denaturation of 3 min at 95°C followed by 20 cycles of 20 s at 95°C, 15 s 55°C, 30 s at 72 and one cycle of final elongation of 1 min at 72°C in a final volume of 50 µl. Qubit™ dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific) was used to measure the concentration of all cDNA samples. The 3’ fragments of the cDNA were released in a restriction reaction using SalI-HH (New England Biolabs) according to the manufacturer’s protocol. The concentration of DNA was measured using Qubit™ dsDNA High Sensitivity Assay Kit and DNA integrity and the size distribution were assessed with High Sensitivity DNA Kit (Agilent Genomics). For HLA target enrichment one TSO-specific universal forward primer and eight gene-specific reverse primers with universal tails for amplicon sequencing were used to amplify exons 1 to 8 in class I genes HLA-A, -B, and -C or exons 1 to 5 in class II genes HLA-DRA, -DRB1, -DPA1, -DPB1, -DQA1 and -DQB1. HLA-A, -B, and -C had one common primer. All seven gene-specific primers were designed to fall within a non-polymorphic region using the known sequence diversity, as described in the international ImMunoGeneTics IMGT/HLA database (http://www.ebi.ac.uk/imgt/hla/). The amplification was performed in 96-well plates with 3 µl of template cDNA, 10× Advantage 2 PCR buffer, 50× Advantage^® 2 Polymerase Mix (Takara, Clontech), 10 mM dNTP (Bioline), 10 µM TSO forward primer and one of the seven HLA gene-specific reverse primers in a total volume of 15 µl. The PCR reaction consisted of an initial denaturation of 30 s at 98°C following three cycles of 10 s at 98°C, 30 s at 55°C, 30 s at 72°C and 27 cycles of 10 s at 98°C, 30 s at 71°C, 30 s at 72°C and final elongation of 5 min at 72°C. To confirm the amplicon lengths and non-specific amplification, 4 samples were selected from each plate with the amplification performed using different gene-specific primer. These samples were run on a 2% agarose gel (Bioline) with 10× BlueJuice™ loading dye (invitrogen by Thermo Fisher Scientific) in 0.5× TBE (Thermo Fisher Scientific) with the GelGreen™ (Biotium) and visualized using the Quick-Load 1kb DNA Ladder (New England Biolabs). DNA of the PCR amplicons was quantified with the Qubit™ dsDNA High Sensitivity Assay Kit and the fragment sizes analyzed with Agilent’s High Sensitivity DNA Kit.