|
| Status |
Public on Jan 08, 2016 |
| Title |
C1_SM1_C39 |
| Sample type |
SRA |
| |
|
| Source name |
iPSC-derived cortical neuron
|
| Organism |
Homo sapiens |
| Characteristics |
iPSc: AH017-7
reprogramming vector: Sendai virus (SeVdp(KOSM)302L)
time: 72 days of neuronal differentiation
cell type: iPSC-derived cortical neuron
|
| Growth protocol |
iPSCs were differentiated into cortical neurons using the Livesey protocol with some modifications (Shi et al. Nat. Protocol. 2012;7(10):1836–1846). In brief, iPSCs were cultured in monolayer on matrigel. Cells were induced using dual SMAD inhibition (1µM dorsomorphin and 10µM SB431542) in neural maintenance media (DMEM/F-12, neurobasal, N-2, B-27, 5µg/ml insulin, 1mM L-glutamine, 100µM non-essential amino acids, 100µM 2-mercaptoethanol, 50 units/ml penicillin and 50mg/ml streptomycin). After the formation of a neuroepithelial sheet, this was passaged into wells plated with L-ornithine and laminin. The subsequent differentiating cells were passaged after different durations in culture to expand cortical neural progenitor stocks. RT-qPCR and immunofluorescence microscopy confirmed the adoption of cortical identity. Cells were treated with 4M cytosine arabinoside for 72 hours prior to single cell analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were dissociated as for single cell RT-qPCR at day 72 of neuronal differentiation. 300,000 DAPI negative cells were sorted into 200L of neural maintenance media. These were loaded onto a small C1 chip according to the Smarter-seq protocol detailed by Fluidigm. Cells were co-stained with Hoechst and propidium iodide. Capture chambers were imaged on an Opera Imaging System.
We continued with lysis, reverse transcription, amplification and library prep in accordance with the Fluidigm Smarter-seq protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
GSE67835_neurons_vs_AH017_7_filtered_expr_iPSC.counts.gz
|
| Data processing |
Trimmomatic with the arguments “LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:50”
Tophat2
Rsubread
DESeq adjustment for library size and ERCC counts from 92 ERCC (External RNA Control Consortium) controls
Genome_build: hg19 plus ERCC 92
Supplementary_files_format_and_content: adjusted counts (tsv)
|
| |
|
| Submission date |
Jun 11, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Adam Handel |
| E-mail(s) |
adam.handel@ndcn.ox.ac.uk
|
| Organization name |
University of Oxford
|
| Department |
Weatherall Institute of Molecular Medicine
|
| Street address |
John Radcliffe Hospital
|
| City |
Oxford |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE69790 |
Single cell transcriptomics analysis of induced pluripotent stem cell-derived cortical neurons reveals frequent dual layer identity |
|
| Relations |
| BioSample |
SAMN03770011 |
| SRA |
SRX1056628 |
