|
| Status |
Public on May 31, 2017 |
| Title |
Sample 26 [CTRL 1 d21] |
| Sample type |
SRA |
| |
|
| Source name |
electrically immature motor neurons
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: electrically immature motor neurons
diagnosis: none (control)
patient id: Patient 4
cell line id: CTRL 1
genotype: Control
days of differentiation: 21
|
| Treatment protocol |
Motor neuron (MN)differentiation was carried out using an adapted version of a previously published protocol [Gupta et al. 2012]. Briefly, iPSCs were first differentiated to neuroepithelium by plating to 100% confluency in chemically defined medium consisting of DMEM/F12 Glutamax, Neurobasal, L-Glutamine, N2 supplement, non essential amino acids, B27 supplement, β-mercaptoethanol (all from Life Technologies) and insulin (Sigma). Treatment with small molecules from day 0-7 was as follows: 1μM Dorsomorphin (Millipore), 2μM SB431542 (TocrisBioscience), and 3μM CHIR99021 (MiltenyiBiotec) for 7 days. At day 8, the neuroepithelial layer was enzymatically dissociated using dispase(GIBCO, 1 mg/ml), plated onto laminin coated plates and next patterned for 7days with 0.5μM retinoic acid and 1μMPurmorphamine. At day 14 spinal cord MN precursors were treated with 0.1μM Purmorphamine for a further 4 days before being terminally differentiated in 0.1 mM Compound E (Enzo Life Sciences) to promote cell cycle exit.
|
| Growth protocol |
Dermal fibroblasts were cultured in OptiMEM +10% FCS medium. The following episomal plasmids were transfected for iPSC generation: pCXLE hOct4 shp53, pCXLE hSK and pCXLE hUL (Addgene), as previously reported [15]. Details of the lines used in this study are reported in Supplementary table 1. Two of the control lines used (control 2 and control 3) are commercially available and were purchased from Coriell (cat. number ND41866*C ) and ThermoFisher Scientific (cat. number A18945) respectively. Induced PSCs were maintained on Geltrex (Life Technologies) with Essential 8 Medium media (Life Technologies) and passaged using EDTA (Life Technologies, 0.5mM). All cell cultures were maintained at 37°Cand 5% carbon dioxide.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells using the Maxwell RSC simplyRNA cells kit (Promega), that includes DNase treatment, according to manufacturer’s instructions.
1 ug of total RNA was subjected to Ribo-Zero treatment TruSeq Stranded mRNA preparation Kit (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence and then mapped to h19 ribosomal RNA using bowtie v1.1.2 with parameters -q -p 8 -v 0. Unmapped reads were finally mapped to hg19 whole genome using tophat2 with parameters -g 1 -p 8 --library-type fr-firststrand.
For differential gene and transcript expression analysis we ran Kallisto and Sleuth. Kallisto was used to 1) build a transcript index from the Ensembl GRC38 release 85 Homo sapiens transcriptome (-k 31), 2) pseudo-align the RNA-seq reads to the transcriptome and 3) quantify transcript abundances (-b 100 --single -l 275 -s 50 --rf-stranded). We next identified differentially expressed transcripts and genes with Sleuth, a companion program that uses Kallisto results to differentiate between true biological expression differences and variation resulting from sources of experimental noise.
Genome_build: hg19
Supplementary_files_format_and_content: Relative transcript abundance and transcript per million as obtained from Kallisto software.
|
| |
|
| Submission date |
Apr 27, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Raphaelle Luisier |
| E-mail(s) |
raphaelle.luisier@gmail.com
|
| Phone |
0041762432198
|
| Organization name |
Idiap Research Institute
|
| Lab |
Genomics and Health Informatics
|
| Street address |
Rue Marconi 19, PO Box 592
|
| City |
Martigny |
| State/province |
Valais |
| ZIP/Postal code |
1920 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE98288 |
Progressive motor neuron pathology and the role of astrocytes in a human stem cell model of VCP-related ALS |
| GSE98290 |
Post-transcriptional remodelling is temporally deregulated during motor neurogenesis in human ALS models |
|
| Relations |
| BioSample |
SAMN06842444 |
| SRA |
SRX2767166 |
