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| Status |
Public on May 03, 2024 |
| Title |
H9FF polCA7_distal (batch with mutant samples of GSE175592) |
| Sample type |
SRA |
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| Source name |
Human embryonic stem cells (hESC) H9 obtained from WiCell
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Human embryonic stem cells (hESC) H9 obtained from WiCell
genotype: wild-type
treatment: POLARIZED WITH 1%FGF8 ORGANIZER EB (OrEB)
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| Treatment protocol |
Fusion with organizer EB at D3 where specified
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| Growth protocol |
Organoids were generated with a modified version of what previously described (Lancaster and Knoblich, 2014; Lancaster et al., 2013). Briefly, on D0 hESCs and iPSCs were treated with accutase to single cell suspention and transferred to an ultra-low binding 96-well plate (9000 cells/well) for rOrgs, or to a PMDS mould (5,5x106 cells/mould) for eOrgs. Cells aggregated into embrioyd bodies in mTeSR1 supplemented with 50 µM Rho-associated protein kinase (ROCK) inhibitor. PMDS moulds have been sterilized with autoclaving and gamma irradiation, then treated with Pluronic F127 (Sigma-Aldrich P2443) 1% in PBS and wash with PBS several times prior to usage. From D1 on, the medium was replaced daily with Neural Induction Medium (NI) containing DMEM-F12 supplemented with 1X N2 supplement, 1ug/ml heparin solution, 1X GlutaMAX, 1X MEM-NEAA and 50 µM ROCK inhibitor. On D7, embryoid bodies were embedded into droplets of Matrigel and transferred into 6-cm dishes containing Imp-A Medium consisting of 50% DMEM-F12, 50% Neurobasal medium, 1X N2, 1X B27 – Vitamin A, 2.5 g/ml Insulin, 0.05mM BME, 1X GlutaMAX, 1X MEM-NEAA and 1X Antibiotic-Antimycotic (Thermo Fisher 15240062). On D15, media was replaced with Imp+A Medium consisting of 50% DMEM-F12, 50% Neurobasal medium, 1X N2, 1X B27, 2.5 µg /ml Insulin, 0.05mM BME, 1X GlutaMAX, 1X MEM-NEAA, 1X Antibiotic-Antimycotic and organoids were moved to on an orbital shaker under 52 rpm rotating speed. The medium was changed every 2-3 days. From D30 onwards, organoids were fed with Imp+A Medium supplemented with 1% (v/v) matrigel basement membrane, BDNF (20 ng/ml) and GDNF (20 ng/ml)
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
All the polCAs and control (conCAs) have been manually dissected at a stereomicroscope and processed for RNA extraction. For bulk RNAseq, samples were lysed in 350 µl of RLT buffer supplemented with βmercaptoehtanol and RNA was extracted using the RNeasy Micro Kit (Qiagen 74004) following the manufacturer’s protocol. For scRNAseq, manually dissected at a stereomicroscope and processed for dissociation with gentleMACSTM Dissociator in Trypsin/Accutase solution with TURBO™ DNase (Thermo AM2238, 2 U/µL). After dissociation, DPBS-/- + 10% FBS has been gradually added to stop reaction. Samples have been then centrifuged at 400G for 5min at 4ºC and supernatant was aspirated without touching the pellet. The pellet has been resuspended in additional 500µl of DPBS-/- + 10% FBS, filtered through FACS tubes and stained for viability dye DRAQ7 (Biostatus, DR70250, 0.3mM). Live cells have been sorted with a BD FACS Aria™ III on Alexa 700 filter and barcoded with unique CMOs for MULTI-seq analysis.
For bulk RNAseq, we then assessed RNA concentration and quality with an RNA 6000 Nano Chip (Agilent Technologies) and enriched for mRNA with NEBNext Poly(A) mRNA Magnetic Isolation Module; barcoded samples were then multiplexed and sequenced 50 bp single-end on a HiSeq 2500 (Illumina). mRNA sample enrichment, library preparation, and sequencing were performed by the VBCF NGS Unit (https://www.vbcf.ac.at).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Basecalling: RTA 1.18.66.3
Adapter have been clipped with trimgalore (v0.5.0)
Abundant sequences (iGenomes hg38) are removed with bowtie2 (v2.3.4.1).
Cleaned reads are aligned against the genome (GRCh38) with STAR (v2.6.0c).
Reads are counted towards their corresponding gene (ENSEMBL v94) with featureCounts (v1.6.2).
Assembly: GRCh38
Supplementary files format and content: feature count output file (tab-separated; last column contains counts)
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| Submission date |
Apr 29, 2023 |
| Last update date |
May 03, 2024 |
| Contact name |
Camilla Bosone |
| E-mail(s) |
camilla.bosone@imba.oeaw.ac.at
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| Organization name |
IMBA
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| Street address |
Dr Bohr gasse 3
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| City |
wien |
| State/province |
Austria |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE231318 |
Specification of a rostro-caudal axis in cortical assembloids through a polarized source of FGF8 (bulk RNA-Seq) |
| GSE231320 |
Specification of a rostro-caudal axis in cortical assembloids through a polarized source of FGF8 |
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| Relations |
| BioSample |
SAMN34441590 |
| SRA |
SRX20144747 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7257027_140940_gene.featureCounts.txt.gz |
4.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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