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Transcriptome analysis of electroporated/GFP+ cells from embryonic mouse cortex E15.5 (E13.5 + 48 h), shRNA against circSlc45a4, mRNA Slc45a4 and scramble KD

ABSTRACT: We perturbed the expression levels of circSlc45a4 and mRNA Slc45a4 in developing mouse cortex (E15.5) by RNAi/shRNAs. 48 h post-electroporation the cells were isolated by FACS (GFP+), RNA was extracted with Trizol and polyA+ libraries were generated with Illumina TruSeq Stranded mRNA LT Sample Prep Kit. We find many significant changes in the transcriptomic profile after knockdown of circSlc45a4 and mRNA Slc45a4, that are distinct from each other.

ORGANISM(S): Mus musculus

PROVIDER: GSE124000 | GEO | 2019/03/31

REPOSITORIES: GEO

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Project description:mRNA transcript levels in embryonic day 15.5 (E15.5), E17.5 and postnatal day 1 (P1) mouse Crb1KOCrb2ΔRPC against mouse Crb1KO neuroretina (Run1), and in E15.5 mouse Crb1KOCrb2ΔRPC against wild type neuroretina (Run2) were analyzed. A comparison between Crb1KOCrb2ΔRPC and Crb1KO retina, at E15.5, or E17.5, or P1 on 100% C57BL/6JOlaHsd, yielded only subtle persistent changes at the transcriptional level over time (Figure 1 G-I, respectively), despite significant differences in morphology. As inter-nal positive control at E15.5, E17.5 and P1 we used ATP-binding cassette, sub-family D (ALD), member 4 (Abcd4), which is substantially upregulated in mouse retina expressing the Cre recombinase fused to the Chx10 promoter, with the Abcd4 gene immediately adja-cent to the Chx10Cre locus in the mouse genome. To further examine potential transcriptional changes at E15.5, we compared Crb1KOCrb2ΔRPC mice in 100% C57BL/6JOlaHsd genetic background against wild type C57BL/6JRccHsd mice. In this setting, we observed 40 differentially expressed genes (DEGs) compared to the previously 0 DEGs (E15.5; adj. p-val < 0.01 & log2FC > 1.5). The Crb1KOCrb2ΔRPC mice on C57BL/6JOlaHsd genetic background, due to mutations in the synuclein alpha (Snca), multimerin-1 (Mmrn1), and Crb1 gene, do not express Snca, Mmrn1, or Crb1 gene transcripts, and express high levels of Abcd4 due to the adjacent Chx10Cre transgene on chromosome 6. We made use of all 4 genes as negative and positive controls in the gene expression profiling, since the mice on C57BL/6JRccHsd genetic background have no mutations in Snca, Mmrn1 or Crb1 and express low levels of Abcd4. Analysis of the E15.5 data revealed upregulated expression of the WD repeat and FYVE domain-containing protein 1 (Wdfy1) gene also known as FYVE domain-containing pro-tein localized to endosomes (Fens-1), which encodes a phosphatidylinositol 3-phosphate binding protein that contains a FYVE zinc finger domain and multiple WD-40 repeat do-mains.

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Transcriptome analysis of electroporated/GFP+ cells from embryonic mouse cortex E15.5 (E13.5 + 48 h), shRNA against circSlc45a4, mRNA Slc45a4 and scramble KD

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