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| Status |
Public on Jan 06, 2020 |
| Title |
GDNF 3 |
| Sample type |
SRA |
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| Source name |
hES cell graft in rodent brain
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
graft type: Ventral midbrain
treatment: GDNF
host: Athymic (Foxn1nu) nude mice
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| Treatment protocol |
Intrastriatal injections of adeno-associated viral vector carrying GDNF (AAV-GDNF) into the striatum 3 weeks prior to, or after 3 weeks after transplantation.
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| Growth protocol |
Human PSCs were seeded on Laminin-521 (10ug/ml; StemCell Technologies) in the presence of dual SMAD inhibitors (SB431542, 10uM, 0-5 days in vitro (DIV), R&D systems; and LDN193189, 200nM, 0-11DIV, Stemgent) to promote neural induction. Sonic hedgehog C25II (100ng/ml; R&D systems) and Purmorphamine (2uM; Stemgent) were supplemented into the media from day 1-7DIV to promote ventralisation, in addition to the caudalizing Wnt agonist, CHIR 99021 (3uM; Stemgent, 3-11DIV). From 13DIV, cells were cultured in a ‘maturation media’ consisting of NBB27 supplemented with GDNF (20ng/ml; R&D systems), BDNF (20ng/ml, R&D system), TGFβ3 (1 ng/ml; PeproTech), DAPT (10 μM, Sigma-Aldrich), ascorbic acid (200nM; Sigma-Aldrich) and dibutyryl cAMP (0.05mM; Tocris Bioscience). At 21 DIV differentiating cultures were dissociated in Accutase (Innovative Cell Technologies) to a single-cell suspension and fluorescent activated cell sorting (FACS) performed on a MoFlo cell sorter (Beckman Coulter, MoFlo to isolate LMX1A-GFP expressing progenitors, as previously described (de Luzy et al., 2018). The FACS-isolated cell fraction was resuspended at 100,000 cells/µL in maturation media containing ROCK inhibitor (Y27632, 40uM, Sigma) and stored on ice until the time of implantation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The striatum (containing the transplant) were rapidly dissected from the host brain and flash frozen. The isolated tissue was homogenised using a TissueLyser LT (Qiagen) and total RNA extracted using the RNeasy Mini Kit (Qiagen) including DNase treatment.
Mixed species cDNA libraries were prepared using TruSeq stranded mRNA sample preparation kit (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Align reads to Human Genome and Output concordant aligned reads as fastq - HiSat2 (v2.0.3) : hisat2 -x "UCSC_hg38" --al-conc "output" --rna-strandness R --no-mixed --no-discordant.
Align concordant human aligned reads to Mouse Genome and output unaligned reads as fastq - HiSat2 (v2.0.3) : hisat2 -x "UCSC_mm10" --un-conc "output" --rna-strandness R --no-mixed --no-discordant.
Align Human Specific Reads to Human Genome and output concordant aligned reads as BAM - HiSat2 (v2.0.3) : hisat2 -x "UCSC_hg38" --rna-strandness R --no-mixed --no-discordant. Output concordant aligned reads as BAM
Generate Human specific read counts for each gene - HTSeq-count (v0.6.1) : htseq-count --mode=union --stranded=reverse --minaqual=255 --type='exon'
Differential Expression Analysis - DESeq2 (v1.12.4) : DESeq (test = "Wald", fitType= "parametric")
Genome_build: Homo_sapiens_UCSC_hg38 & Mus_musculus_UCSC_mm10
Supplementary_files_format_and_content: tab-delimited text files containing feature counts (htCount)
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| Submission date |
Jan 05, 2020 |
| Last update date |
Jan 07, 2020 |
| Contact name |
Christopher Bye |
| Organization name |
The Florey Institute of Neuroscience and Mental Health
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| Lab |
Personalised Theraputics
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| Street address |
University of Melbourne
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| City |
Melbourne |
| State/province |
Victoria |
| ZIP/Postal code |
3010 |
| Country |
Australia |
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| Platform ID |
GPL16512 |
| Series (1) |
| GSE143136 |
Viral Delivery Of GDNF Promotes Functional Integration Of Human Stem Cell Grafts In Parkinson’s Disease |
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| Relations |
| BioSample |
SAMN13736456 |
| SRA |
SRX7502302 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4250838_GDNF-3_TSStrmRNA_L000.tab.gz |
103.9 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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