Single cell sequencing of adrenocortical tumor tissue utilizing the scFAST-seq method

Single-cell RNA sequencing (scRNA-seq) is a technology that allows for the analysis of gene expression at the level of individual cells. In many cases, droplet microfluidics are used for preparing DNA libraries. SeekOne DD scFAST-seq from Beijing SeekGene employs semi-random primers, offering advantages in detecting transcripts, particularly non-polyadenylated transcripts and regions that are distant from the polyadenylated RNA tail. The introduction of new strategies in scRNA-seq represents a promising approach for research, such as studying the properties of adrenocortical tumors. This study aims to investigate the biological properties of adrenocortical tumor samples by SeekOne scFAST-seq methodology. Single cells were isolated from tissue samples using ACME HS followed by cryopreservation in 3xSSC*10% DMSO and methanol cell fixation. Key adaptations of the ACME HS method were the supplement of solution composition with 0.1M NAC and the introduction of additional washing steps in cold high salt 3xSSC* buffer. ACME dissociation was conducted in ~ 1 hours on a rotator at room temperature, with periodic pipetting of the solution. Afterward, we removed the ACME solution and washed the pellet with a two-step washing in cold 3xSSC. Single cells were captured, barcoded, and cDNA libraries were generated using the V1.1-SeekOne DD Single Cell Full-length RNA Sequence Transcriptome-seq Kit (SeekGene) according to the manufacturer’s instructions. Final libraries were multiplexed and sequenced on an Illumina Novaseq 6000 platform, using the S4 Reagent Kit v1.5.