Multiplex Barcoding for Single-Cell Sequencing

For both single-cell DNA and RNA experiments, the last round of BAG barcode was added in the same way. Many strategies can be used to perform split-and-pool (Cao et al. 2017 (link); Vitak et al. 2017 (link); Rosenberg et al. 2018 (link); Cao et al. 2019 (link)), and additional rounds of BAG split-barcodes can be added based on the common sequence from the previous round (Supplemental Method S2). Here we used PCR to add 96 different BAG barcodes in the last split. In each well, there is a universal PCR primer and a well-specific primer containing different barcodes. BAGs from the last split-and-pool were evenly distributed into 96 wells. DNA BAGs were amplified using NEBNext ultra II Q5 master mix (NEB M0544). RNA BAGs were amplified using KAPA HiFi HotStart ReadyMix (Roche KK2602). The PCR product was pooled together and purified using AMPure XP magnetic beads (Bechman Coulter A63881).