GSM3611805: C1 Day23; Homo sapiens; RNA-Seq
Organoids were collected by scraping cells from a petri dish (10 cm diameter) and dissociated using cold active protease (Bacillus licheniformis) as described by Adam et al., 2017 (Adam et al., 2017). About 20 organoids generated in 3D culture under each condition were dissociated with 5 mg of cold active protease (Sigma, #P5380) in 1 mL DPBS for 15 min on a slow moving shaker with repeated trituration at 4°C to avoid high temperature artifacts. Cells were passed through a 30 µm cell strainer, washed once in DPBS supplemented with 0.04% BSA, centrifuged at 300 x g for 10 min and adjusted to a concentration of 200,000 cells/mL in 0.04% BSA in DPBS. We followed the DropSeq protocol described by Macosko et al. with some modifications described in detail by Menon et al (Macosko et al., 2015; Menon et al., 2018). Consistent with our previous findings, in the NPC stage (Figure 8A) ~ 500,000 cells were isolated from the standard condition while only ~200,000 cells were detected under the glutamine-deprived condition. Approximately 100,000 cells of each group were loaded onto a DropSeq device for single cell transcriptomic analysis. DropSeq protocol described by Macosko et al. Next generation sequencing of generated scRNA-Seq libraries was performed on an Illumina HiSeq2500 platform (paired end 26x115 bp). R1 is the barcode and R2 is the mRNA sequence
GEO Accession
GSM3611805