The tdTomato-YFP double-positive cells, tdTomato-positive cells, and YFP-positive cells were isolated by FACS. Single cells were captured with the Chromium System (10x Genomics) following the manufacturer’s recommendation. Briefly, sorted cells were washed with PBS containing 0.04% BSA and loaded into a Chromium Next GEM Chip G with reagents of Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (10X Genomics). The Cell-Gel Beads in Emulsion were generated and incubated to generate the barcoded cDNA. cDNA was cleaned with Dynabeads (10x Genomics) and amplified with 12 cycles of PCR. cDNA was then enzymatically fragmented, end-repaired, poly-A–tailed, adapter-ligated, and amplified by PCR. The constructed libraries were sequenced on an Illumina HiSeq 2500/4000 platform (150 bp paired-end reads).
The FASTQ file from the sequencing was processed by Cell Ranger v4.0.0 (10x Genomics). For alignment, we made a custom reference package including YFP and tdTomato with mouse genome (mm10). Cellranger count was used for read alignment and gene expression quantification.
The FASTQ file from the sequencing was processed by Cell Ranger v4.0.0 (10x Genomics). For alignment, we made a custom reference package including YFP and tdTomato with mouse genome (mm10). Cellranger count was used for read alignment and gene expression quantification.