Fig. 1
Deletion of the TERRA-20q locus markedly affects TERRA expression. a Scheme depicting the WT and the CRISPR-deleted allele for the 20q-TERRA locus located in the subtelomere of the chromosome 20, q-arm. The position of the gRNAs (E1 and S2) and the primers used to genotype the deletions are also shown. The black arrows represent the primers to amplify the CRISPR-deleted allele and the white arrows the ones to amplify the WT allele located inside the 20q-TERRA locus. b Ethidium bromide gels showing the WT and the CRISPR-deleted allele for the 20q-TERRA locus detected by PCR in a WT cellular pool and in different clones of the U2OS cells. c RNA from the a WT cellular pool, WT expanded clones (#1 and 2), or 20q-TERRA KO clones (#A7, E1, E6, E8, and H8) from the USOS cell line was isolated and used for TERRA detection by RNA dot-blot with a probe against the TERRA-UUAGGG-tract; 18S serves as loading control. (Graph) TERRA quantification normalized by 18S (mean values ± s.e.m., n = 3 biological replicates). d Representative confocal microscopy images of RNA-FISH against TERRA-UUAGGG-tract (green) in the U2OS WT clones (#1 and 2) and in the 20q-TERRA KO clones (#A7, E1, E6, E8, H8, and C4). Scale bar, 10 μm. (Graph) Quantification of the total spot intensity per nucleus normalized by nucleus area (mean values ± s.e.m., n = cells analyzed). One-way ANOVA with Dunnett’s post test was used for the statistical analysis (*p < 0.05, **p < 0.01, and ***p < 0.001)
