normal rat kidney fibroblasts  (ATCC)

Journal: iScience

Article Title: Recruitment of transcriptional effectors by Cas9 creates cis -regulatory elements and demonstrates distance-dependent transcriptional regulation

doi: 10.1016/j.isci.2025.114040

Figure Lengend Snippet: Cardiac-specific open putative regulatory elements are distributed around cardiac genes (A) A density histogram of CM-specific open chromatin distribution around the TSS of 221 highly enriched cardiac genes, showing high density of cardiac-specific open chromatin very close to the TSS with nonlinear decay at greater distances (Kbp). (B) Same data as in (A) presented as a map where the 221 loci were centered around the highly enriched cardiac genes TSS and the distribution of CM-specific open chromatin was plotted in a window of ±100 Kbp. (C) Analysis of expression of six selected highly enriched cardiac genes in cardiomyocytes (blue) and fibroblasts (green) using DE-seq2 normalized RNA-seq counts on a logarithmic scale, showing two orders of magnitude higher expression in cardiomyocytes for these genes ( n = 3 for cardiomyocytes and n = 2 for fibroblasts, black bars show the mean expression). (D) Heatmap showing the major cardiac transcription factors are very lowly expressed in fibroblasts. Data from RNA-seq with each column representing a different biological sample ( n = 6 and 4 for cardiomyocytes and fibroblasts, respectively. See also .

Article Snippet: Rat fibroblasts (Rat2, CRL-1764) and human fibroblasts (HEK293, CRL-1573) were acquired from ATCC and grown with culture medium.

Techniques: Expressing, RNA Sequencing

Journal: iScience

Article Title: Recruitment of transcriptional effectors by Cas9 creates cis -regulatory elements and demonstrates distance-dependent transcriptional regulation

doi: 10.1016/j.isci.2025.114040

Figure Lengend Snippet: Recruitment of activation and repression domains controls gene expression in a distance-dependent manner from multiple genomic sites in human cells (A) Repression maps shown as multi-track diagrams in two cardiomyocyte-specific gene loci (MYBPC3 and MYH7), spanning 150 Kbp each. The TSS of each gene is shown in red arrows. Tracks showing (from top to bottom): the genomic track with exons and introns in blue; repression showing index gene % repression in iPSC-CM following Ad-dCas9-KRAB targeting versus non-targeting sgRNA control, measured by RT-qPCR normalized to GAPDH. ENCODE ATAC-seq and H3K27ac-seq tracks in hiPSC are shown in blue and red, respectively. Scale of repression 0%–100% in square brackets, average % repression from each sgRNA site in black bars, red dots over black bars represent SEM, only bards with two-tailed t-test p < 0.05 versus control are shown, n = 3 biological replicates. (B) Activation maps in human HEK293 fibroblasts shown as multi-track diagram of 3 CM-specific genes (TNNI1, COX6A2, and RCAN1) spanning 150 Kbp each. The TSS of each gene is shown in red arrows. Tracks showing (from top to bottom): the genomic track with exons and introns in blue, fold change activation following dCas9-VPR targeting versus non-targeting sgRNA control, measured by RT-qPCR normalized to GAPDH. ENCODE DNAse and H3K27ac-seq tracks in fibroblast cells are shown in blue and red, respectively. The scale for activation for the track is shown in square brackets, average fold activation for each sgRNA site in black bars, and red dots over black bars indicate SEM. All black bars have two-tailed t-test p < 0.05 versus control, n = 3 biological replicates.

Article Snippet: Rat fibroblasts (Rat2, CRL-1764) and human fibroblasts (HEK293, CRL-1573) were acquired from ATCC and grown with culture medium.

Techniques: Activation Assay, Gene Expression, Control, Quantitative RT-PCR, Two Tailed Test