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| Status |
Public on Sep 06, 2020 |
| Title |
RNAseq in EB-Day4[D0+RA+SAG][noDox(iFlag.Hoxc6)]_replicate3 |
| Sample type |
SRA |
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| Source name |
ES-derived embryoid bodies+4D RA+SAG
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| Organism |
Mus musculus |
| Characteristics |
strain genotype/variation: Ainv15(iFlag.Hoxc6)
treatment: EB + 4 Days RA + SAG, +48h NO Dox
cell type: Embryoid Bodies
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| Treatment protocol |
Embryoid bodies (EBs) were obtained by plating trypsinized (Gibco) mESCs in AK medium (Advanced DMEM/F12:Neurobasal (1:1) medium (Gibco), 7% KnockOut SR (vol/vol) (Gibco), 2 mM L-glutamine, 0.1 mM ß-mercaptoethanol and penicillin–streptomycin (Gibco),) at 37 °C, 5% CO2 (day -2). On day 0, EBs were split 1:2 and AK medium was replenished and supplemented with 1 μM all-trans retinoic acid and 0.5 μM smoothened agonist (SAG) (Millipore, 566660). TF induction was performed by adding 3 μg/mL of Doxycycline (Sigma, D9891) on day 2. For RNA-seq experiments, 3.5x10^5 mESCs were plated in 100mm suspension dishes (Corning).
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| Growth protocol |
mESC lines were cultured in 2-inhibitors based medium (Advanced DMEM/F12:Neurobasal (1:1) medium (Gibco), supplemented with 2.5% ESC-grade fetal bovine serum (vol/vol, Corning), N2 (Gibco), B27 (Gibco), 2mM L-glutamine (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1000 U/mL leukemia inhibitory factor (Millipore), 3μM CHIR (BioVision) and 1 μM PD0325901 (Sigma)) on 0.1% gelatin (Millipore) coated plates at 37 °C, 8% CO2.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were collected prior to TF induction (day 2 of RA/SAG differentiation) and 48h after Doxycycline (Dox) treatment. RNA was extracted by using TRIzol LS Reagent (Life Technologies) and purified using the RNAeasy mini kit (Qiagen). Agilent High Sensitivity RNA Screentape (Agilent, 5067-5579) was used to check RNA integrity.
500ng of RNA was used to prepare RNA-seq libraries and spiked-in with ERCC Exfold Spike-in mixes (Thermo Fisher, 4456739). RNA-seq libraries were prepared using TruSeq Stranded mRNA Library Preparation kit (Illumina, 20020594). Library size was verified using High Sensitivity DNA ScreenTape (Agilent, 5067-5584). The KAPA library amplification kit was used on Roche Lightcycler 480 for library quantification before pooling.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
HV3T2BGX7_n01_mb162
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| Data processing |
Basecall conversion to fastq files was performed using Picard (v 2.8.2)
Alignment: Fastq files obtained from RNA-sequencing were aligned to the genome using the splice-aware STAR (v2.7.0c) (Spliced Transcripts Alignment to a Reference) aligner (Dobin & Gingeras, 2016).
Computing Abundance: Mapped reads were assigned to NCBI RefSeq annotated mm10 genes using the featureCount function in Rsubread (v1.30.9) (Liao, Smyth, & Shi, 2019). RefSeq genes with matching Entrez IDs were merged into a single gene by Rsubread. Following read summarization, read counts were normalized using the ‘rlog’ or regularized log transformation in DESeq2 (Love, Huber, & Anders, 2014) (v1.20.0).
Differential Expression Analysis: The log2 fold change (LFC) in gene expression levels between iHox motor neuron vs. progenitor motor neurons was estimated using DESeq2. A q-value < 0.01 and LFC > 2 was used to define differentially expressed genes between progenitor motor neurons, noDox controls, iHoxc6 MNs, iHoxc8 MNs, iHoxc9 MNs, and iHoxc10 MNs.
Genome_build: mm10
Supplementary_files_format_and_content: Unnormalized Read Counts at mm10 annotated genes
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| Submission date |
Dec 19, 2019 |
| Last update date |
Sep 06, 2020 |
| Contact name |
Shaun Mahony |
| E-mail(s) |
mahony@psu.edu
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| Phone |
814-865-3008
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| Organization name |
Penn State University
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| Department |
Biochemistry & Molecular Biology
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| Lab |
Shaun Mahony
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| Street address |
404 South Frear Bldg
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| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE142378 |
Diversification of posterior Hox patterning by graded ability to engage inaccessible chromatin [RNA-seq] |
| GSE142379 |
Diversification of posterior Hox patterning by graded ability to engage inaccessible chromatin |
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| Relations |
| BioSample |
SAMN13635339 |
| SRA |
SRX7416210 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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