GSM8061311: hPSC-derived Monocyte/Macrophages, Day 28, scRNA-seq; Homo sapiens; RNA-Seq
For Day 35 HIOs with and without Macrophages and Day 28 Macrophages: Organoid were washed with cold Cell Recovery solution and minced in HBSS with Mg2+ and Ca2+ 1% BSA. Original supernatants collected and spun down and readded into the sample. HIOs were mechanically and enzymatically digested enzymes from Neural Tissue Dissociation Kit as recommended by Manufacturer's instructions for 45-60 minutes at 37C. Samples were shaken and fluxed through a P1000 then P200 tip every 10 mins. Dissociated cells were washed and filtered through a BSA coated 70um follow 40um cell strainer, centrifuged at 300 RPM for 5 minutes, and resuspended in 2% FBS in HBSS with y-27632 and 1% BSA. Day 28 Macrophages were subjected to the same mechanical and enzymatic manipulations as the dissociated HIOs. Library was performed according to the manufacter's instructions (single cell Chromium 3' v3 GEM protocol, 10x Genomics) loading 12,800 cell for approximately 8,000 captured cells. Briefly, cells 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.