GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM3528719 Query DataSets for GSM3528719
Status Public on Dec 24, 2018
Title RNA-Seq of Mus musculus: EN-KO+Hoxa1 rep2
Sample type SRA
Source name embryo, blastocyst embryo, blastocyst
Organism Mus musculus
Characteristics genotype/variation: HoxA1 enhancer-KO + Hoxa1
cell type: Embryonic stem cell
strain: 129/Ola
treatment: RA induced differentiation
cell line: E14
Treatment protocol For RA treatment, ESCs were induced to differentiate by LIF/2i withdrawal and addition of 2uM RA (Sigma). Culture medium was replaced every day.
Growth protocol Mouse ESCs were grown in culture dishes coated with 0.1% gelatin in Glasgow Minimum Essential Medium (GMEM) supplemented with 15% fetal bovine serum (FBS), 100nM nonessential amino acids, 1% sodium pyruvate, 200mM glutamate, 1% penicillin- streptomycin, 50uM β-mercaptoethanol, 10ng/mL LIF, 1uM PD0325901 (a MEK inhibitor) and 3uM CHIR99021 (a GSK inhibitor). The medium was replaced every 2-3 days (32,33)
Extracted molecule total RNA
Extraction protocol 1. For RNA -seq: cells were lysed with Trizol reagent (Life Technologies) and RNA was extracted according to the manufacturer's instructions. 2. For Capture-C:Briefly, cells were fixed with 1% (vol/vol) formaldehyde for 10 min at room temperature, quenched with 125 mM glycine in PBS, and then lysed in cold lysis buffer (10 mM Tris–HCl, pH7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 0.2% NP-40, 1× complete protease inhibitor cocktail (Roche)). Chromatin was digested with DpnII (New England Biolabs) at 37°C overnight. Fragments were then diluted and ligated with T4 DNA ligase (Takara) at 16°C overnight. Cross-linking was reversed by overnight incubation at 60°C with proteinase K (Bioline). Then 3C libraries were purified by phenol-chloroform followed by chloroform extraction and ethanol-precipitated at −80°C overnight. Sequencing libraries were prepared from 10μg of the 3C library by sonication to an average size of 200 ~300 bp and indexed using NEBnext reagents (New England Biolabs), according to the manufacturer's protocol. Enrichment of 2μg of an indexed library incubated with 3uM of a pool of biotinylated oligonucleotides (probe sequences are listed in Supplementary Table 7) was performed using the SeqCap EZ Hybridization reagent kit (#05634261001, Roche/NimbleGen), following the manufacturer's instructions. Two rounds of capture employing 48~72 and 24 hr hybridizations, respectively, were used. Correct library size was confirmed by agarose gel electrophoresis, and DNA concentrations were determined using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific). All sequencing was performed on Hi-Seq 2500 platforms using paired 150 bp protocols (Illumina).
The Capture-C library was constructed with NEBNext DNA Library Prep Master Mix Set for Illumina (NEB).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model HiSeq X Ten
Description E14_RNA_seq_htseq_gene_count_martix.xls
Data processing RNA-seq analysis: Clean reads were mapped to Ensemble mm10 using Hisat2 with default parameters. Gene reads were counted by Htseq. Fold changes were computed as the log2 ratio of normalized reads per gene using DEseq2 R package.
Capture-C analysis: The analysis of Capture-C processing step referred to previously reported procedures (Davies, J.O., Telenius, J.M.2016 Nature methods).
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include genes count for each Sample and wig
Submission date Dec 23, 2018
Last update date Dec 24, 2018
Contact name Dianhao Guo
Organization name Nankai University
Street address 94 Weijin Road
City Tianjin
ZIP/Postal code 300071
Country China
Platform ID GPL21273
Series (1)
GSE124306 A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation
BioSample SAMN10618145
SRA SRX5173552

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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