Thyroid tissuewere cut into about 1-2mm3 pieces and digest in SoloTM Tumor Dissociation Kit (Sinotech Genomics, JZ-SC-58201) at 37°C for 30-60min. Then we stopped enzymatic digestion with excess RPMI-1640 medium, and filtered cell with 40 μm Cell strainer. Single cell solution were kept on ice before loading to BD Rhapsody cartridge for single cell transcriptome capturation. Cells were firstly stained with two fluorescent dye, Calcein AM and Draq7, for precisely determination of cell concentration and viability via BD Rhapsody™ Scanner (BD Biosciences) before single cell multiplexing labelling. Cell of each sample were sequentially labeled with BD Human Single-Cell Multiplexing Kit (Cat. No. 633781) which utilizing the cell-hashing technology [1] mainly to provide higher sample throughput and eliminate batch effect for single cell library preparation and sequencing. Briefly, cells from each sample were labeled by antibodies with different sample tags respectively and washed twice with Stain Buffer (FBS) (BD Biosciences, Cat. No. 554656) before pooling all samples together. Pooled samples were loaded in one BD Rhapsody micro-well cartridge based on Fan et al [2]. Cell capture beads were then loaded excessively to ensure that nearly every micro-well contains one bead, and the excess beads were washed away from the cartridge. After lysing cells with lysis buffer, Cell capture beads were retrieved and washed prior to performing reverse transcription. Microbeads-captured single cell transcriptome and SampleTag molecules were generated into cDNA library and SampleTag library separately containing cell labels and unique molecular identifiers (UMI) information