title: GSM7421800: W8_donor2_SOX10_plus_iMicroglia; Homo sapiens; RNA-Seq
library_construction_protocol: For the enzymatic cellular dissociation, 30 organoids, for each experiment, were first washed in Hank's balanced salt solution (HBSS) and, then, incubated with a freshly prepared dissociation solution (HBSS, papain and DNAase I) for 45 min at 37°C on an orbital shaker (90 rpm), and gently triturated using a P1000 pipette. Cells were incubated again for 15 min at 37°C on an orbital shaker (90 rpm) centrifuged and gently triturated using a P1000 pipette. After the single cell dissociation, the cell suspension was mixed (1:1) with 10 mg/mL ovomucoid protease inhibitor solution and centrifuged at 300g for 5 min. The pellet was resuspended in 1X PBS containing BSA (1:50). Cells were counted using trypan blue and a haemocytometer under bright field. A yield of 700–1,200 cells per μL per sample was targeted. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, GCs 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 a sample index were added.