Single-cell RNA-seq of telencephalic organoids from healthy CTRL and HD (Huntington's disease) hESC (RUES2) grown as mono-culture or co-culture of chimeric organoids
Single-cell RNA-seq of telencephalic organoids from healthy CTRL and HD (Huntington's disease) hESC (RUES2) grown as mono-culture or co-culture of chimeric organoids
Additional protocol information can be found in the Addtional Files. The organoids were transferred in a dish to cut away the excess of Matrigel with a blade and then were processed by dissociation using Papain Dissociation System with DNase (Worthington, LK003150) following the manufacturer's recommendations. The incubation time was adjusted, based on size, at 20 min in the orbital shaker at 37°C. Then the samples were centrifugate for 1min at 1000 rpm and resuspended in DMEM+Neurobasal containing N2 supplement (1:100, Gibco), B27 supplement (1:50, Gibco), and Rock Inhibitor Y-27632 (10µM, provided by CHDI Foundation, CHDI-00197406-0001-007). For the dissociation into single cell suspension, the samples were pipetted up and down first with 1000µl pipettes and following with glass pipettes of increasingly smaller tip diameter (fire-polished). The suspension was filtered with disposable filter of 20µm (Celltrics sysmex, 04-004-2325). The cells were counted with Countess Automated Cell Counter to check viability (higher than 90%) and concentration. The suspension was centrifugated for 1 min at 1000 rpm and resuspended at the concentration of 1x104 cells /µl in PBS containing 0.04% weight/volume BSA (Ambion, AM2616, 400 µg/ml). The Chromium Single Cell 3′ Library & Gel Bead Kit v2 (PN- 120237), Chromium Single Cell 3′ Chip kit v2 (PN-120236) and Chromium i7 Multiplex Kit (PN-120262) were used according to the manufacturer's instructions in the Chromium Single Cell 3′ Reagents Kits V2 User Guide The Chromium Single Cell 3′ Library & Gel Bead Kit v2 (PN- 120237), Chromium Single Cell 3′ Chip kit v2 (PN-120236) and Chromium i7 Multiplex Kit (PN-120262) were used according to the manufacturer's instructions in the Chromium Single Cell 3′ Reagents Kits V2 User Guide