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Sample GSM2316548 Query DataSets for GSM2316548
Status Public on Sep 30, 2016
Title 26Dp1_B08_smart-seq
Sample type SRA
 
Source name cultured embryonic stem cells
Organism Homo sapiens
Characteristics days in culture: 26
cre line: DCX+
differentiation#: RM165049
control: FALSE
Growth protocol 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.
Extracted molecule polyA RNA
Extraction protocol 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
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Data processing 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.
Genome_build: hg19
Supplementary_files_format_and_content: RSEM TPM gene expression matrix
 
Submission date Sep 15, 2016
Last update date May 15, 2019
Contact name Zizhen yao
E-mail(s) zizheny@alleninstitute.org
Organization name Allen Institute for Brain Science
Department MAT
Street address 551 N 34th St #200
City Seattle
State/province WA
ZIP/Postal code 98103
Country USA
 
Platform ID GPL16791
Series (2)
GSE86894 REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS
GSE86982 REGION-SPECIFIC NEURAL STEM CELL LINEAGES REVEALED BY SINGLE-CELL RNA-SEQ FROM HUMAN EMBRYONIC STEM CELLS [Smart-seq]
Relations
BioSample SAMN05779220
SRA SRX2170330

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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