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Sample GSM2590532 Query DataSets for GSM2590532
Status Public on May 31, 2017
Title Sample 27 [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
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
BioSample SAMN06842442
SRA SRX2767167

Supplementary file Size Download File type/resource
GSM2590532_27.txt.gz 2.0 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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