title: GSM8822089: LS foreskin, biol rep 2; Homo sapiens; RNA-Seq
library_construction_protocol: The procedure initiated with the fastidious creation of a single-cell suspension, succeeded by a rigorous assessment of its viability. It was ascertained that the cells exhibited a remarkable degree of activity, surpassing the 85% threshold, and were distinguished by their excellent uniformity and minimal impurities. To augment the efficiency of isolating individual cells, the cell concentration was carefully fine-tuned to an exact range of 700 to 1200 cells per microliter. During the construction of the single-cell RNA sequencing (scRNA-seq) library, each cell, along with its associated cellular enzymes, was attached to a gel bead. This assembly was subsequently encapsulated within an oil droplet, forming a single-cell gel bead-in-emulsion (GEM). Each gel bead was uniquely labeled with a distinct barcode, incorporated a unique molecular identifier (UMI) sequence, and was equipped with a poly-dT primer sequence designed to initiate reverse transcription. The GEM droplets were then collected in Eppendorf tubes, where cell membranes were lysed using specifically designed recovery reagents, releasing the mRNA. The liberated mRNA interacted with the enzymes, dNTP substrates, and the poly-dT primer sequence within the droplets. Under precisely controlled conditions in a polymerase chain reaction (PCR) apparatus, reverse transcription was initiated, resulting in the synthesis of complementary DNA (cDNA). In the final step, the cDNA underwent PCR amplification, thereby completing the construction of the scRNA-seq library.