Fig. 6

Screening of eQTLs in the Sall2 KO congenic region. a Pipeline based on the linear dependency of gene expression to genotype for the detection of expression quantitative trait loci (eQTL) in congenic regions. Digital counts of gene expression from congenic DEGs in WT, heterozygous and KO genotypes are normalized (e.g. RUVSeq package, available in Bioconductor) and used for further analysis. Then, lineal regression of gene counts across genotypes is obtained, previously defining arbitrary values for each genotype (WT = 1, Het = 0.5, KO = 0). Genes with slopes significantly different than zero are selected according to P < 0.05. Finally, an output list of genes with candidate eQTLs is obtained. b Left: DEGs with significant eQTLs in control after linear regression analysis as described in (A). Genes with slope significantly different from zero are candidate eQTLs and potential modifier genes in the Sall2 KO. We settled the P = 0.05 threshold using a dashed line. Right: Same analysis for the congenic DEGs with doxorubicin perturbation. We settled the P = 0.05 threshold using a dashed line. We selected candidate DEGs with eQTLs based on significant P-values both in control and with doxorubicin perturbation. Four genes were selected for further analysis (Ang, Tmem260, 4930579G18Rik, and Osgep, denoted as red dots in both graphs). c qPCR validation of Ang expression in Sall2 WT and KO MEFs with or without doxorubicin treatment (16 h with 1 μM doxorubicin). Shown are Ang expression levels normalized to Polr2A gene when compared to levels in WT or doxorubicin treatment. (N = 4; data is represented as means ± s.e.m.). *** P < 0.001 versus WT or doxorubicin treatment; Student’s T-test. d Left: Normalized reads per kilobase per million (RPKM) of Angiogenin (Ang) against RNA Pol II (Polr2A) across the eight mice founder strains. Right: Same analysis as described for the Rnase4 gene. e Sashimi plots of the Ang/Rnase4 gene expression in C57BL/6J (upper) and 129S1/SvlmJ (lower) strains. Per-base expression is plotted on the y-axis of Sashimi plot; genomic coordinates on the x-axis and the gene structure are represented on the bottom (in blue, obtained from the USCS server). Lower black arrows indicate Ang expression and upper black numbers indicate Ang junctions. Lower black numbers indicate Rnase4 junctions. f Ratio of Ang versus Rnase4 junctions in strains with (129S1/SvlmJ, A/J, NOD/ShiLtJ, NZO/HILtJ, PWK/PhJ and WSB/EiJ, respectively) and without (C57BL/6J and CAST/EiJ strains) SNPs in the Ang/Rnase4 locus. g Left: Representative Western blot for SALL2, ANG, and ACTIN in Sall2 WT and KO iMEFs. Right: Quantification of ANG protein bands normalized with ACTIN in the bar graph (N = 3, *** P < 0.001 versus WT; Student’s T-test.). h Left: The same analysis of (G) for ANG in scramble (shCtrl) and Sall2-silenced cells (shSall2). Right: Quantification of ANG protein bands normalized with ACTIN in the bar graph at the right (N = 3, *** P < 0.001 versus shCtrl; Student’s T-test.). i Validation of Rnase4 levels in the shRNA-silencing model of Sall2 in iMEFs. Shown are the expression levels normalized to RNA pol II (Polr2A gene) for Rnase4 when compared to levels in shCtrl. We expressed the values as fold change from shCtrl (N = 3, * P < 0.05 versus shCtrl; Student’s T-test). j Validation of Ang levels in a CRISPR-Cas9 silencing model of Sall2 in iMEFs. We expressed the values as fold change from control CRISPR (N = 3, * P < 0.05 versus Control CRISPR; Student’s T-test). k Luciferase assay with the murine Ang/Rnase4 promoter electroporated in Sall2 WT and null iMEFs. We measured luciferase and β-galactosidase activities. R.L.U = relative luminescence units to β-gal (N = 3, *** P < 0.001 versus WT; Student’s T-test)