2025-06-05
ENA first public
ERC000011
ENA-CHECKLIST
Medical Research Center Oulu and Research Unit of Clinical Medicine, University of Oulu and Oulu University Hospital
INSDC center name
public
INSDC status
ERS20253459
SRA accession
E-MTAB-14174:Sample 11
Submitter Id
ArrayExpress
broker name
primary cell line
cell_line
mouse embryonic stem cell
cell_type
not collected
collection date
house mouse
common name
Protocols: Mouse ESC cultures were established at the Biocenter Oulu Transgenic and Tissue Phenotyping Core Facility (Oulu, Finland) using the 2i method (Nichols et al., 2009) from four homozygous NHLRC2 p.Asp148Tyr, four Nhlrc2 KO, and four WT littermates.   Embryos were isolated on E2.5 from super-ovulated female oviducts. The embryos were cultured overnight in KSOM (Merck, Darmstadt, Germany) supplemented with glycogen synthase kinase-3 (CHIR 99021, Axon Medchem, Groningen, the Netherlands) and MAP kinase kinase 1 (PD 0325901, Axon Medchem, Groningen, the Netherlands) inhibitors (2i). The embryos were transferred to the 2i medium (N2B27 + ESGRO + 2i) until they developed into the blastocyst stage. The trophectoderm of the blastocyst was removed through immunosurgery (Solter & Knowles, 1975), and the inner cell mass was transferred first onto feeders in 2i medium and then after the Accutase treatment (Accutase: Gibco™ StemPro™ Accutase™ Fisher Scientific) on gelatinized (Gelatin, Sigma-Aldrich, St. Louis, MO) plates in 2i medium supplemented with 100 μ/ml penicillin–streptomycin (Sigma-Aldrich, St. Louis, MO).  The ES cells were transferred to gelatinized (Millipore, Billerica, MA) plates, and the medium was changed to a complete basal medium (Millipore, Billerica, MA) supplemented with a 2i Supplement Kit (Millipore, Billerica, MA) and 100 μ/ml penicillin–streptomycin (Sigma-Aldrich, St. Louis, MO). The medium was changed daily, and Accutase (Millipore, Billerica, MA) was used for passaging the cells. After passage four, the cells did not attach to the gelatinized plate and were cultured in suspension thereafter. The cells were harvested from a 10-cm cell culture dish and RNA isolation was performed directly after.  RNA isolation from mESCs was performed using RNeasy Mini Plus Kit (Qiagen, Hilden, Germany) and RNase-Free Dnase Set (Qiagen, Hilden, Germany) according to manufacturer’s instruction. Precise concentrations were measured with Qubit-assay and Bioanalyzer-assay were used for determining the RNA integrity.  Library was prepared using RNA seq, TruSeq® Stranded Total RNA kit (Illumina, San Diego, CA, USA).
description
mouse embryo stage
developmental stage
Healty control
disease
wild type genotype
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
inner cell mass
organism part
Mus musculus
scientific_name
C57BL/6NCrl
strain
Sample 11
title