GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM4226473 Query DataSets for GSM4226473
Status Public on Sep 06, 2020
Title RNAseq in EB-Day4[D0+RA+SAG][D2+48hDox(iFlag.Hoxc6)] replicate 2
Sample type SRA
Source name ES-derived embryoid bodies+4D RA+SAG with induced transcription factors
Organism Mus musculus
Characteristics strain genotype/variation: Ainv15(iFlag.Hoxc6)
treatment: EB + 4 Days RA + SAG, +48hours Dox
cell type: Embryoid Bodies
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).
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.
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.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description HV3T2BGX7_n01_mb155
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
Submission date Dec 19, 2019
Last update date Sep 06, 2020
Contact name Shaun Mahony
Phone 814-865-3008
Organization name Penn State University
Department Biochemistry & Molecular Biology
Lab Shaun Mahony
Street address 404 South Frear Bldg
City University Park
State/province PA
ZIP/Postal code 16802
Country USA
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
BioSample SAMN13635337
SRA SRX7416212

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap