GSM8645351: Neurabin I+/- Neurons, rep1; Homo sapiens; RNA-Seq
Neural progenitor cells were generated from human episomal iPSC line (Thermo Fisher Scientific Cat. No.: A18945) by inducing the neural ectoderm for 7 days using neural induction media (NIM, Thermo Fisher Scientific). For this purpose, 5 x 105 cells/mL iPSCs were seeded in a six-well plate precoated with rhLaminin-521 (2.5 lg/mL concentration in DPBS with calcium, magnesium) in the presence of 10 uM Rho kinase inhibitor at 37 C in 5% CO2 incubator. The next day (day 1), the ectoderm formation was induced by replacing culture media with NIM consisting of 1x neurobasal media supplemented with 1x neural induction supplement. The spent media was replaced with fresh media every other day until day 7. On day 8, NIM was removed, and NPCs were maintained in neuronal expansion media (NEM) until 80% confluency. NEM was prepared by mixing 1x neurobasal media and 1x DMEM/F12 KO media supplemented with 0.5x neural induction supplement. NPCs were differentiated into neurons by maintaining the cells in neuronal differentiation medium (NDM) for 15 days. Briefly, after harvesting NPCs, 1 x 106 cells/mL were seeded in NEM on a culture vessel precoated with 50 lg/mL poly-Dlysine and 100 lg/mL rh-Laminin-521 in the presence of 10 lM Rho kinase inhibitor and incubated overnight at 37C in a 5% CO2 incubator. The next day (i.e., day 12), the spent NEM was replaced with fresh prewarmed NDM (1x neurobasal medium and 1x DMEM/F-12 supplemented with 1x B27 supplement, 1x N2 supplement, 10 ng/mL bFGF, and 10 mM GlutaMAX supplement). The media was changed every other day until day 22. The newly differentiated iPSC-derived neurons were used for experimental analysis after days 27?30. The data was analyzed using scRNAseq. iPSC-derived NPCs and neurons (day 30 of differentiation) were dissociated using TrypLE Express and washed twice with PBS to remove culture media and strained using 70- lm cell strainers. Finally, the cell pellet was resuspended in PBS þ 0.04% BSA and diluted to achieve 1,200 cells/lL with a target recovery of 10,000 cells. The cell suspension was then subjected to generating barcoded single stranded cDNA molecules using a 10x chromium platform following the manufacturer?s protocol. Barcoded cDNAs (10 ng) were conjugated to Biotin tags and amplified for 13 cycles using LongAmp Hot Start Taq. Later, M280 strepta vidin beads were used to pull down the full-length bar coded cDNA molecules, followed by adapter ligation and sequencing using the PromethION system from Oxford Nanopore Technologies (Oxford, UK). For both NPCs and neurons, 50 fmol barcoded cDNA library was loaded to an independent PromethION flow cell (R10.4.1) for every sample. The average N50 of the mRNA long isoform sequencing was 900 bp