Platinum multiplex pcr master mix

PCR reactions (10 μl total) consisted of 2.5 μl (0.5× final) Platinum® Multiplex PCR Master Mix (Applied Biosystems), 1.8 μl 25 mM MgCl₂ (4.5 mM final), 1.5 μl forward lambda reaction product (non-purified), 1.5 μl reverse lambda reaction product (non-purified), 2 μl cDNA, and 0.7 μl nuclease-free water (not DEPC-treated). The reaction cycle profile was as follows: initial denaturation at 95 °C for 5 min; 22 cycles of 95 °C for 30 s, 60 °C for 3 min, 72 °C for 60 s; and final extension at 68 °C for 10 min. Unit PCR reaction of genotyping assays was 20 μl, with the same concentration of reagents, and 18 cycles of PCR. Unit PCR reaction of transcriptomics experiments was 10 μl, with cycle numbers between 16 and 22.

SOE-PCR analysis was performed using Illumina primers: P5: 5′ AAT GAT ACG GCG ACC ACC GA 3′ and P7: 5′ CAA GCA GAA GAC GGC ATA CGA GAT 3′. FAM-conjugated probe 3Taq1: 5′ AGA TCG GAA GAG CGT CGT GTA GG 3′ was targeted to the homology domain shared by the antibody and cell barcodes. qPCR was performed using Platinum Multiplex PCR Master Mix (Thermo Fisher Scientific), 2% w/v Tween 20, 2% w/v PEG 6000 and Mx3005P qPCR System (Agilent Technologies). Drops were generated in HFE 7500 fluorinated oil (3M) containing 2% PEG-PFPE amphiphilic block copolymer surfactant (Ran Biotechnologies). Prior to PCR reaction oil exchanged was performed using fluorinated oil (FC40) with 5% PEG-PFPE amphiphilic block copolymer. PCR parameters as follows: 95 °C for 2 min, 11 cycles of 95 °C for 30 seconds, 60 °C for 90 seconds, 72 °C for 20 seconds followed by final extension at 72 °C for 10 min. Droplets were coalesced by adding 100 μL perfluorooctanol (Sigma, 370533) and centrifuging at 1000 g for 1 minute. The aqueous phase was transferred to a spin column for purification (Zymo Research, DNA Clean & Concentrator).

A total of 33 Y-STR markers were amplified by combining 22 Y-STR markers of the PowerPlex^® Y23 System (Promega Corporation) and 11 Y-STR markers of an adapted RM-Yplex assay (13 Y-STRs in total) based on Alghafri et al. [26 (link)]. The modified primer mix can be found in Supplementary File S1. Instead of the DYS518 marker that was used by Alfghafri et al. [26 (link)], the marker DYS464 was added to the multiplex PCR. This multi-copy Y-STR marker with at least four copies on the Y chromosome was selected for its outstanding diversity [27 ,28 (link)]. The primer sequence for DYS464 was taken from Redd et al. [29 (link)] and labelled with ATTO 550. PCR amplification was performed in a final volume of 15 µL per reaction: 7.5 µL Platinum™ Multiplex PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), 3.3 µL primer mix according to Supplementary File S1, 3.2 µL PCR grade water, and 1 µL DNA template (1 ng/µL). To confirm a duplication observed within the marker Y-GATA-H4 in one sample we performed an additional PCR using the Yfiler™ Plus PCR Amplification Kit (Thermo Fisher Scientific) with this sample and the sample of the associated son. CE ana­lysis was carried out on the 3500 Genetic Analyzer (Applied Biosystems, Forster City, CA, USA) and data were analysed using the GeneMapper IDX version 1.4 software (Thermo Fisher Scientific).