biostudies-arrayexpressAnniina HiltunenMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14174Fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA) (OMIM 618278) is a rare infantile-onset disease caused by variants in NHL repeat containing 2 (NHLRC2) gene. NHLRC2 is essential for mouse gastrulation (Hiltunen et al., 2022). Here, RNA sequencing was performed on four wild type (WT), four homozygous C57BL/6NCrl-Nhlrc2em1Rthl (FINCA disease causing variant) (Hiltunen et al., 2020), and four homozygous C57BL/6NCrl-Nhlrc2tm1a(KOMP)Wtsi (Nhlrc2 knock out) mouse embryonic stem cell lines each established from individual mouse embryos by the 2i method (Nichols et al., 2009). FINCA variant mESCs express the full length endogenous Nhlrc2 mRNA modified to containing a c.442G > T variant leading to p.Asp148Tyr as well as a silent c.408C>A for genotyping purposes (Hiltunen et al., 2020). In the Nhlrc2 KO mESCs the transcription is halted by a trapping cassette between exons 4 and 5 (Hiltunen et al., 2022; Skarnes et al., 2011). References Hiltunen, A. E., Kangas, S. M., Ohlmeier, S., Pietilä, I., Hiltunen, J., Tanila, H., McKerlie, C., Govindan, S., Tuominen, H., Kaarteenaho, R., Hallman, M., Uusimaa, J., & Hinttala, R. (2020). Variant in NHLRC2 leads to increased hnRNP C2 in developing neurons and the hippocampus of a mouse model of FINCA disease. Molecular Medicine, 26(1), 123. https://doi.org/10.1186/s10020-020-00245-4 Hiltunen, A. E., Vuolteenaho, R., Ronkainen, V. P., Miinalainen, I., Uusimaa, J., Lehtonen, S., & Hinttala, R. (2022). Nhlrc2 is crucial during mouse gastrulation. Genesis, 60(3), e23470. https://doi.org/10.1002/dvg.23470 Nichols, J., Silva, J., Roode, M., & Smith, A. (2009). Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo. Development (Cambridge), 136(19), 3215–3222. https://pubmed.ncbi.nlm.nih.gov/19710168/ Skarnes, W. C., Rosen, B., West, A. P., Koutsourakis, M., Bushell, W., Iyer, V., Mujica, A. O., Thomas, M., Harrow, J., Cox, T., Jackson, D., Severin, J., Biggs, P., Fu, J., Nefedov, M., de Jong, P. J., Stewart, A. F., & Bradley, A. (2011). A conditional knockout resource for the genome-wide study of mouse gene function. Nature, 474(7351), 337–342. https://doi.org/10.1038/nature10163biostudies-arrayexpressSequencing - Each sample was sequenced across three lanes (L001-L003). The sequencing was performed in pair-end mode with NextSeq 550 High Output v2.5 Kit (150 cycles).Library Construction - Library was prepared using RNA seq, TruSeq® Stranded Total RNA kit (Illumina, San Diego, CA, USA).Nucleic Acid Extraction - 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.Sample Collection - 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.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Before starting the analysis, the multiple lanes were merged to generate a “master” fastq file per sample. The data was analysed using Chipster (Kallio et al., 2011). Quality control for the fastq files was performed using FastQC (Wingett & Andrews, 2018). Alignment was performed using HISAT2 (D. Kim et al., 2015) for paired end reads with reference genome Mus_musculus.GRCm38.95. Alignment quality control was performed with RseQC (Wang et al., 2012). HTSeq was used for quantification (Anders et al., 2015).MetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 550RNA-seq of total RNAMus musculusAltered behaviour and immune response in mice with NHLRC2 p.Asp148Tyr variant.Anniina HiltunenReetta HinttalaHiltunen AE, Kangas SM, Gondane A, Koivisto H, Salokas K, Heikkinen A, Salo MH, Röning T, Tallgren A, Glumoff V, Denis MC, Karagianni N, Myllyharju J, Varjosalo M, Tanila H, Itkonen HM, Rämet M, Uusimaa J, Hinttala R.falseRNA-seq of mouse embryonic stem cells with knockout of Nhlrc2 gene or addition of FINCA disease causing NHLRC2 p.Asp148Tyr variantFibrosis, neurodegeneration, and cerebral angiomatosis (FINCA) (OMIM 618278) is a rare infantile-onset disease caused by variants in NHL repeat containing 2 (NHLRC2) gene. NHLRC2 is essential for mouse gastrulation (Hiltunen et al., 2022). Here, RNA sequencing was performed on four wild type (WT), four homozygous C57BL/6NCrl-Nhlrc2em1Rthl (FINCA disease causing variant) (Hiltunen et al., 2020), and four homozygous C57BL/6NCrl-Nhlrc2tm1a(KOMP)Wtsi (Nhlrc2 knock out) mouse embryonic stem cell lines each established from individual mouse embryos by the 2i method (Nichols et al., 2009). FINCA variant mESCs express the full length endogenous Nhlrc2 mRNA modified to containing a c.442G > T variant leading to p.Asp148Tyr as well as a silent c.408C>A for genotyping purposes (Hiltunen et al., 2020). In the Nhlrc2 KO mESCs the transcription is halted by a trapping cassette between exons 4 and 5 (Hiltunen et al., 2022; Skarnes et al., 2011). References Hiltunen, A. E., Kangas, S. M., Ohlmeier, S., Pietilä, I., Hiltunen, J., Tanila, H., McKerlie, C., Govindan, S., Tuominen, H., Kaarteenaho, R., Hallman, M., Uusimaa, J., & Hinttala, R. (2020). Variant in NHLRC2 leads to increased hnRNP C2 in developing neurons and the hippocampus of a mouse model of FINCA disease. Molecular Medicine, 26(1), 123. https://doi.org/10.1186/s10020-020-00245-4 Hiltunen, A. E., Vuolteenaho, R., Ronkainen, V. P., Miinalainen, I., Uusimaa, J., Lehtonen, S., & Hinttala, R. (2022). Nhlrc2 is crucial during mouse gastrulation. Genesis, 60(3), e23470. https://doi.org/10.1002/dvg.23470 Nichols, J., Silva, J., Roode, M., & Smith, A. (2009). Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo. Development (Cambridge), 136(19), 3215–3222. https://pubmed.ncbi.nlm.nih.gov/19710168/ Skarnes, W. C., Rosen, B., West, A. P., Koutsourakis, M., Bushell, W., Iyer, V., Mujica, A. O., Thomas, M., Harrow, J., Cox, T., Jackson, D., Severin, J., Biggs, P., Fu, J., Nefedov, M., de Jong, P. J., Stewart, A. F., & Bradley, A. (2011). A conditional knockout resource for the genome-wide study of mouse gene function. Nature, 474(7351), 337–342. https://doi.org/10.1038/nature101632025-05-22T00:00:00Z2025-06-17T09:00:51.883Z2024-06-14T17:32:54.594ZE-MTAB-14174ERP161129EFO_0002944EFO_0004170EFO_0009653EFO_0005518EFO_0003816EFO_0004184