GSM9035978: hPSC-derived HAGO+SM, Day 37, biol rep 1, diff 1; Homo sapiens; RNA-Seq
For in vitro Day 37 HAGOs and Day 35 HIOs: Organoid were washed with cold Cell Recovery solution and minced in HBSS with Mg2+ and Ca2+ 1% BSA. Organoids 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 HBSS with Y-27632 and 1% BSA. For hAGO+SM TXP: Organoid was removed from mouse kidney capsule after 10 weeks in vivo growth. The organoid was separated from surrounding kidney and mechanically minced into small pieces using scapel in HBSS with Mg2+ and Ca2+. HAGO TXP 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. 400 ul of 10x TrypLE was added for the last 20 min of enzymatic digestion. 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 HBSS with Y-27632 and 1% BSA. The scRNA-seq assay was performed according to the manufacter's instructions (single cell Chromium 3' v3.1 GEM protocol, single or dual indexed, 10x Genomics) loading 12,800 cell for approximately 8,000 captured cells on either a Chromium or Chromium X instrument. 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 and amplified using 14 cycles by PCR.  Full-length, barcoded cDNA was then enzymatically fragmented, sized-selected using SPRIselect reagent, adapter-ligated, and amplified for library construction. During the library construction, Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences, and sample indexes were added. Samples were pooled and run on the NovaSeq 6000 with S4 flow cells using the following sequencing parameters:  R1: 28 cycles, i7: 8 cycles, i5: 0 cycles, R2: 91 cycles (HAGO 1, HAGO+SM, HAGO+SM+NCC 1, HAGO+SM+NCC 2) or R1: 28 cycles, i7: 10 cycles, i5: 10 cycles, R2: 90 cycles (HAGO 2, HIO, HAGO+SM TXP).