6-Plex human/mouse species-mixing experiment

HEK293T and NIH3T3 cells were prepared 1 day before Drop-Seq and plated on six-well plates (Techno Plastic Products, Switzerland) at approximately 70% confluency. Transfection of SBO (28 pmol/ml) was performed 4 hours before Drop-Seq, as described above. All cell samples were trypsinized using trypsin-EDTA (0.25%) and phenol red (Gibco, USA), pooled together, and washed four times with phosphate-buffered saline (PBS; Gibco, USA). The cells were then resuspended in 0.01% bovine serum albumin (BSA) + PBS, passed through a 40-μm filter, counted using the LUNA Automated Cell Counter (Logos Biosystems, Korea), and diluted at a final combined concentration of 400 cells/μl. The diluted sample library was run once in Drop-Seq, and sample preparation and sequencing were performed as above. From one Drop-Seq run, about 77,000 beads were obtained and divided into 24 PCRs for cDNA amplification. Sample preparation was completed using two reactions of the Nextera XT DNA Library Preparation Kit (Illumina, USA).