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Sample GSM4273609 Query DataSets for GSM4273609
Status Public on Jan 17, 2020
Title C9KO-1_1
Sample type SRA
Source name iPSC-derived motor neurons
Organism Homo sapiens
Characteristics genotype: hexanucleotide repeat expansion in intron of C9ORF72 gene as well as a knockout of the start codon in exon 2
Treatment protocol not applicable
Growth protocol Neurons were cultured in N2B27 medium with 200 μM ascorbic acid, 10 ng/ml BDNF, and 20 ng/ml GDNF, 200 μM dbcAMP, 1 ng/ml TGFβ3
Extracted molecule total RNA
Extraction protocol Total RNA was isolated using Qiagen RNeasy columns including a column DNase digest
mRNA was isolated from total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturer’s instructions. Final elution was done in 15ul 2x first strand cDNA synthesis buffer (NEBnext, NEB). Samples were then directly subjected to the workflow for strand specific RNA-Seq library preparation (Ultra Directional RNA Library Prep II, NEB). For ligation custom adaptors were used 1: (Adaptor-Oligo 5'-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3', Adaptor-Oligo 2: 5'-P-GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC-3'). After ligaton adapters were depleted by an XP bead purification (Beckman Coulter) adding the beads solution in a ratio of 1:1. Indexing was done during the following PCR enrichment using custom amplification primers carring the i7 index sequence indicated with ‘NNNNNNNN’. (Primer1: AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T, primer2: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNNNN GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T. After two more XP beads purifications (1:1) libraries were quantified.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Data processing bcl2fastq: 2.19.1
alignment: Processed fastq files were aligned with the aligner GSNAP (v. 2017-11-15) to the human reference hg38 from Ensembl with the support of splice site detection which was done with the Ensembl annotation (gtf file) version 87
genecount: Fragments were assigned to Gene with featureCounts (v1.5.3) and Ensembl annotation (gtf file) version 87 was used to find gene regions
Genome_build: hg38
Supplementary_files_format_and_content: bfx1299.genecounts.csv is a tab separated file which is the featureCounts output. The first columns contain gene information such as Ensembl ID, Strand, Length, Start and End. Afterwards the 9 samples are listed
Submission date Jan 15, 2020
Last update date Jan 17, 2020
Contact name Jared Sterneckert
Organization name TU Dresden
Department Center for Molecular and Cellular Bioengineering (CMCB)
Lab iPS Cells and Neurodegenerative Disease
Street address Fetscherstraße 105
City Dresden
State/province Saxony
ZIP/Postal code 01307
Country Germany
Platform ID GPL18573
Series (1)
GSE143743 Knocking out C9ORF72 exacerbates axonal trafficking defects associated with hexanucleotide repeat expansion and reduces levels of heat shock proteins I
BioSample SAMN13869632
SRA SRX7568595

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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