GSM7401927: FD, NHEM, scRNAseq; Homo sapiens; RNA-Seq
Fsk daily and alternate day NHEMs were trypsinized and spun down at 400g then washed and resuspended in 1x DPBS and taken for library preparation Libraries was prepared according to the manufacter's instructions (single cell 3' v3.1 (dual index) protocol, 10x Genomics). Briefly, NHEMs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and two sample indexes were added.