|
| Status |
Public on Jan 17, 2020 |
| Title |
C9-1_1 |
| Sample type |
SRA |
| |
|
| Source name |
iPSC-derived motor neurons
|
| Organism |
Homo sapiens |
| Characteristics |
genotype: untargetted cells containing hexanucleotide repeat expansion in intron of C9ORF72
|
| 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 |
| E-mail(s) |
Jared.Sterneckert@tu-dresden.de
|
| 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 |
|
| Relations |
| BioSample |
SAMN13869612 |
| SRA |
SRX7568589 |
