Evaluation of single cell classifiers for single cell RNA-seq datasets

Three human cell lines, K562, HEK293T and A431, and one murine cell line, L929, are mixed with equal proportion in two experiments. One mixture, named Mix3, consists of K562, HEK293T and L929. The other, named Mix4, consists of K562, HEK293T, A431 and L929. These cell lines were processed with the Singleron GEXSCOPETM protocol using GEXSCOPETM Single Cell RNA Library Kit (Singleron) and sequenced on an Illumina Xten. FASTQ data were preprocessed using fastp and STAR. Three human cell lines, K562, HEK293T and A431, and one murine cell line, L929, were cultured separately in DMEM (Thermo Fisher Gibco) with 10% fetal bovine serum (FBS) (Thermo Fisher Gibco), and 1% penicillin-streptomycin (Beyotime Biotechnology ) in an incubator with 5% carbon dioxide at 37°C. Single cell suspension with 1×105 cells/mL in concentration in PBS (HyClone) were prepared. Two experiments were conducted. One is named as Mix3 , where K562, 293T and L929 cell suspensions were mixed in 1:1:1 ratio. The other is named as Mix4 , where cell suspensions of all four cell lines were mixed in 1:1:1:1 ratio. Mixed single cell suspensions were then loaded onto microfluidic devices and scRNA-seq libraries were constructed following Singleron GEXSCOPETM protocol using GEXSCOPETM Single Cell RNA Library Kit (Singleron). Sequencing was performed on Illumina Xten with 150 bp paired end reads to obtain a sequencing depth of approximately 6.5K reads/cell.