GSM8493748: Liver-36; Homo sapiens; RNA-Seq
Frozen samples were dissociated on a GentleMACS in 1mL of TST buffer (https://www.nature.com/articles/s41591-020-0844-1). Nuclei were counted and resuspended in PBS+BSA+RNAse inhibitor before loading on a 10x 3' v3.1 channel. Library construction was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, after nuclei loading and gel bead-in-emulsion (GEM) formation, 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 a sample index were added.