GSM7696724: hPSC-derived HCO, Day 22, biol rep 1, scRNA-seq; Homo sapiens; RNA-Seq
For Day 22 and Day 37 HCOs and Day 14 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. HCOs 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 14 Macrophages were subjected to the same mechanical and enzymatic manipulations as the dissociated HCOs. For Day 28 HCOs Hematopoietic Floaters: Supernatants of Day 28 HCOs was collected, spun down, and resuspended in HBSS with BSA and did not undergo enzymatic digestion. For HCO TXP: Organoid was removed from mouse kidney capsule after 15 weeks in vivo growth. The organoid was separated from surrounding kidney capsule and outer most muscle layer, opened, and flushed the lumen of mucus. HCOs were shaken in 5mL TryPLe + 5uL y-27632 for 60 minutes at 4C. 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 PBS without Mg++ or Ca++ with y-27632. Cells were stained with Sytox Blue viability dye and sorted on a FACS Aria cell sorter and 100,000 live cells were collected in 5mL PBS with 5% FBS, 0.5% BSA, 5uL y-27632. 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.