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Sample GSM7906146 Query DataSets for GSM7906146
Status Public on Jan 24, 2024
Title patient_#79787_iPSC_3
Sample type SRA
 
Source name cell line
Organism Homo sapiens
Characteristics tissue: cell line
cell line: #79787
cell type: iPSC
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_E7_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

Supplementary data files not provided
Raw data are available in SRA

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