GSM8594821: mix_CSF.MS_48h; Homo sapiens; RNA-Seq
For the enzymatic cellular dissociation, 30 organoids, for each experiment and for each donor, 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 resuspend ed 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. For each condition, cell viability was assessed using Luna II Automated Cell Counter. Samples with more than 80% viable cells were processed for downstream analysis. For droplet-based scRNAseq, libraries were prepared using the Chromium Single Cell 3ʹ v3, according to the manufacturer's protocol (10x Genomics) without modifications, targeting 10,000 cells per sample. Using the Illumina Novaseq platform, all samples were sequenced at 50,000 reads per cells. Preparation of cDNA libraries and sequencing support were performed at the Center for Omics Sciences (San Raffaele Scientific Hospital, Milan). 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.