|
| Status |
Public on Jan 24, 2024 |
| Title |
patient_#114107_nonpAstro_3 |
| Sample type |
SRA |
| |
|
| Source name |
cell line
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: cell line
cell line: #114107
cell type: nonpAstro
genotype: patient
replicate: 3
|
| Growth protocol |
Briefly, confluent iPSC cultures were dissociated with collagenase, collected with a cell scraper and cultured in suspension to form embryoid bodies. Cells were cultured in mTESR1 with 1× mTESR1 supplement and 10 μM Rock Inhibitor Y-27632 for 24 hours. Then, medium was changed to Astrocyte Medium (AM) supplemented with 20ng/ml Noggin and 10ng/mL PDGFAA for the next two weeks and an additional week with only PDGFAA. Embryoid bodies were then manually dissociated by pipetting and the resulting pAstros were plated on poly-L-ornithine (PLO) and laminin-coated dishes in AM supplemented with 10ng/ml bFGF and 10ng/ml EGF. Upon reaching ~80% confluency, pAstros were passaged using Accutase. pAstros were maintained in culture until ~day 45-50 of glial differentiation and subsequently terminally differentiated into nonpAstros in AM supplemented with 10ng/ml LIF. Media was changed every second day.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells at iPSC, pAstro and nonpAstro stages were collected and RNA was isolated on column using the PicoPureTM RNAextraction kit (Applied Biosystems). RNA quality and concentration were evaluated with an Agilent BioAnalyzer 2100 (Agilent). All samples had a RIN > 9
10 ng of RNA from each sample was used to generate the RNA-seq libraries using bulk-adapted mcSCRB-seq protocol (Bagnoli et al., 2018): cDNA was generated by oligo-dT primers containing well-specific (e.g. sample specific) barcodes and unique molecular identifiers (UMIs). Unincorporated barcode primers were digested using Exonuclease I (Thermo Fisher)
cDNA was pre-amplified using KAPA HiFi HotStart polymerase (Roche) and pooled before Nextera libraries were constructed from 0.8 ng of pre-amplified cleaned up cDNA using Nextera XT Kit (Illumina). 3’ ends were enriched with a custom P5 primer (P5NEXTPT5, IDT) and libraries were size selected using 2% E6 Gel Agarose EX Gels (Life Technologies), cut out in the range of 300–800 bp, and extracted using the Monarch DNA Gel Extraction Kit (New England Biolabs)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Description |
E3V7_S1_H6_m_STAR_hg38
allStar_res_both.csv
|
| Data processing |
Aligment STAR-2.5.3a, hg38, ensembl90
Assembly: hg38, ensembl90
Supplementary files format and content: CSV file with raw data per line
|
| |
|
| Submission date |
Nov 17, 2023 |
| Last update date |
Jan 24, 2024 |
| Contact name |
Giacomo Masserdotti |
| E-mail(s) |
giacomo.masserdotti@helmholtz-munich.de
|
| Organization name |
Helmholtz Zentrum
|
| Department |
ISF
|
| Street address |
Groshaderner Strasse 9
|
| City |
Planegg-Martinstied |
| ZIP/Postal code |
82152 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18460 |
| Series (2) |
| GSE248120 |
Unfolded protein Response is a major hurdle in direct neuronal reprogramming of human astrocytes [Bulk RNA-seq] |
| GSE248129 |
Unfolded protein Response is a major hurdle in direct neuronal reprogramming of human astrocytes |
|
