GSM8515198: CTNNB1_WT_20wk; Homo sapiens; RNA-Seq
Organoids were dissociated into single cells utilizing the Worthington Papain Dissociation System kit, following a previously described protocol (https://protocolexchange.researchsquare.com/article/pex-258/v1) with the following modifications. 2-8 organoids were used per cell line per experiment (Supplemental Table 15). Organoids from the same cell lines were dissociated together so that cells from all organoids of the same cell line were pooled for individual experiments. For 16 week control and 20 week ASD organoids, the organoids were removed from culture medium and washed once with PBS. A clean razor blade was then used to cut individual organoids into approximately four to six pieces. These organoid pieces were then washed 3x with PBS in an attempt to remove dead and/or stressed cells from the interior of the organoid. After the third wash, PBS was removed, papain/DNase solution was added, and the organoid pieces were cut into smaller pieces to start the dissociation process as described in (https://protocolexchange.researchsquare.com/article/pex-258/v1). Upon dissociation completion, cells were diluted in PBS with 0.04% BSA (Millipore Sigma) and kept on ice. Immediately after dissociation was completed, single-cell RNA libraries were generated with the Chromium Next GEM Single Cell 3' Reagent Kit, v3.1 (10X Genomics) following the manufacturer's instructions. For each experiment, 10,000 cells were targeted for recovery.