|Public on Sep 30, 2016
|cultured embryonic stem cells
|days in culture: 26
cre line: DCX-
|hESCs were seeded for a 12-day cortical induction phase in NIM media with SMAD inhibitors and cyclopamine; reseeded for the progenitor expansion phase at D12 and D19 in neural stem cell culture media with EGF and bFGF, and finally at D26 for neural differentiation with neurogenic/neurotrophic factors BDNF, GDNF, NT3, and cAMP. The protocol was ended at D54.
|To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase (ThermoFisher) at 37°C. Light trituration using a P1000 pipette was done every 5 min until nearly all clumps had been dissociated (up to 1 h). Cell suspension was washed and filtered through a 40 μm cell strainer. Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI. Single-cell suspensions were loaded onto a FACSAria II SORP (Becton Dickinson) and sorted directly into PCR strip tubes or plates held in chilled aluminum blocks. Doublets and dead cells were excluded based on forward scatter, side scatter and DAPI fluorescence. Sorting was done using the 130 μm nozzle with the sort mode set to single cell. Accuracy of single-cell sorts was confirmed by sorting DAPI-stained fixed cells onto a dry well of a 96-well plate and analyzing by fluorescence microscopy.
SmartSeq2 protocol. After reverse transcription and template switching, we amplified cDNA with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 22 cycles for RNA from single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) on an Enspire plate reader (PerkinElmer). We used 1 ng of cDNA to generate RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads
|Illumina HiSeq 2500
|Read were aligned transcriptome by RSEM to RefSeq gene annotation (April 23, 2013), and unmapped reads were aligned to genome by Bowtie. The remaining reads were aligned to ERCC.
Supplementary_files_format_and_content: RSEM TPM gene expression matrix
|Sep 15, 2016
|Last update date
|May 15, 2019
|Allen Institute for Brain Science
|551 N 34th St #200
|REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS
|REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq]