|
| Status |
Public on Oct 01, 2021 |
| Title |
Embryonic Stem Cell dervived Hypothalamus-like Neurons (RNA-Seq) Replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
WiCell
|
| Organism |
Homo sapiens |
| Characteristics |
esc line: Hypothalamic Neuron
age (differentiation protocol): 27
sample origin: human ESC H9
|
| Treatment protocol |
ESC were untreated. Upon confluency, cells were cultured in ESC medium without FGF-Basic and Y-27632, but supplemented with Shh (100 ng/ml), Purmorphamine (2 μM), 10 μM SB431542, and 2.5 μM LDN193289. From days 5 to 8, ESC medium was gradually replaced with neuroprogenitor medium (DMEM/F-12 supplemented with 0.1 mM MEM Non-Essential Amino Acids, N-2 Supplement, 0.2 μM ascorbic acid, 0.16% glucose). At day 9, cells were switched into neuronal differentiation medium (DMEM/F-12 supplemented with 0.1 mM MEM Non-Essential Amino Acids, N-2 Supplement, B-27 Supplement minus Vitamin A, 0.2 μM ascorbic acid, 0.16% glucose containing 10 μM DAPT). On day 12, cells were collected with TrypLE Express Enzyme at 37°C for 7 min and washed twice including filtration through pre-wetted 40 μm Corning Sterile Cell Strainer. The hypothalamic progenitor cell pellet was then resuspended with neuronal differentiation medium containing 10 μM Y-27632 for plating on 148 cm2 dishes coated with Poly-L-Ornithine solution (0.01%) and laminin (4 μg/ml) at a seeding density of 16 million cells/148 cm2. After 4 hours (once the cells had completely attached to the plate), the medium was changed to neuronal differentiation medium supplemented with 10 μM DAPT. On day 15, the neuronal differentiation medium was supplemented with 20 ng/ml BDNF (Peprotech Cat # 450-02) until collection on day 27.
|
| Growth protocol |
Human ESC H9 (W09; WiCell) were seeded on Matrigel plates (16 million cells/148 cm2) in DMEM KO medium supplemented with 15% KnockOut Serum Replacement, 0.1mM MEM Non-Essential Amino Acids, 2mM GlutaMAX, 0.06 mM 2-Mercaptoethanol) with FGF-Basic (20 ng/ml media), and 10 mM Rock inhibitor.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from each sample using TRIzol Reagent. RNA was then purified using the Direct-zol RNA Miniprep Kit and depleted of contaminating genomic DNA using DNAse I. Purified RNA was then checked for quality on the Bioanalyzer 2100 using the Nano RNA Chip and samples with a RIN number above 7 were used for RNA-seq library synthesis.
RNA samples were depleted of rRNA using the QIAseq FastSelect RNA Removal Kit then processed into libraries using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina according to manufacturer’s instructions. Quality and quantity of the libraries was measured using the Bioanalyzer 2100 DNA chip and Qubit Fluorometer. Libraries were pooled and sequenced on the NovaSeq 6000 platform using paired-end 51bp reads at the Center for Spatial and Functional Genomics at CHOP.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
total RNA - rRNA depleted
HypothalamusESC_rnaseq_tpm_perReplicate.csv
HPvsHN.DE_rnaseq_edgeR.txt
ESCvsHN.DE_rnaseq_edgeR.txt
|
| Data processing |
Bases were called using Illumina bcl2fastq2 conversion v2.29
Paired-end sequences were aligned to reference genome using STAR (v2.6.0c)
Read counts were calculated using HTSeq-count (v0.6.1). with parameter settings -f bam -r pos -s reverse -t exon -m intersect. Reads were mapped to a curated annotation consisting of GencodeV19 with lincRNA and sno/miRNA annotation from the UCSC Tabler Browser.
Genes located on mitochondiral genomes or annotated as rRNA were excluded from analysis
Differential analysis was performed using edgeR (v3.16.5). Raw reads per genes features were converted to CPM (read Counts Per Million mapped reads). The gene features with median value of less than 0.7 CPM (10 ~ 18 reads per gene feature) across all samples were filtered from differential analysis. The trimmed mean of M-values (TMM) method were used to calculate normalization scaling factors. The differentially expressed genes between ESCs, HPs, and HNs were identified with thresholds of FDR < 0.05 and absolute log2FC > 1.
TPM values were calculated for all genes
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample ...
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| |
|
| Submission date |
Jun 09, 2020 |
| Last update date |
Apr 20, 2022 |
| Contact name |
Matthew C Pahl |
| E-mail(s) |
pahlm@email.chop.edu
|
| Phone |
5704282303
|
| Organization name |
Children's Hospital of Philadelphia
|
| Department |
Center for Spatial and Functional Genomics
|
| Lab |
Struan Grant and Andrew Wells
|
| Street address |
3615 Civic Center Blvd
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE152097 |
Cis-regulatory architecture of human ESC-derived hypothalamic neuron differentiation aids in variant-to-gene mapping of relevant common complex traits (RNA-Seq) |
| GSE152098 |
Cis-regulatory architecture of human ESC-derived hypothalamic neuron differentiation aids in variant-to-gene mapping of relevant common complex traits |
|
| Relations |
| BioSample |
SAMN15186485 |
