title: RNA-seq of mouse embryonic stem cells with knockout of Nhlrc2 gene or addition of FINCA disease causing NHLRC2 p.Asp148Tyr variant
design: RNA-seq of mouse embryonic stem cells with knockout of Nhlrc2 gene or addition of FINCA disease causing NHLRC2 p.Asp148Tyr variant
library_name: Sample 5_p
library_construction_protocol: 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).
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-14174:Sample 5_p