Diabetes mellitus is caused by either insulin resistance or insulin deficiency. The pancreatic beta cells are the primary producers of insulin. Large-scale CRISPR screens combined with single-cell RNA sequencing (scRNA-seq) on beta cells has identified novel insulin regulators and revealed the presence of a highly complex inner network. Here, we performed pooled CRISPR delivery with single-cell transcriptome analysis on the MIN6 cell line, a pancreatic beta-cell line. We have presented the scRNA-seq readout and demonstrated that the MIN6 cell line might develop genetic heterogeneity with increasing passage number based on GO and KEGG pathway analysis. Both computational and biological analyses revealed that the function of MIN6 cell lines could be divided into five clusters, including endocrine cells, basal cells, epithelial cells, and neuroendocrine cells. The fifth cluster was different from the other four clusters due to the differentially expressed insulin transcription and was called the lncRNA-enriched cluster. The experiments also confirmed that uncharacterized lncRNAs GM26917 and Cenpw were associated with insulin transcription. This study provides information that can be used to systematically characterize insulin regulator genes and other genes that control protein folding and vesicle trafficking.