pcdna5 frt to mvenus 3xflag gateway  (Addgene inc)

Journal: bioRxiv

Article Title: A dual role for CTCF in development

doi: 10.64898/2026.03.17.712290

Figure Lengend Snippet: Effect of CTCF depletion on gene expression during gastruloid development A. Schematic representation of the RNA-seq timecourse experiment performed on CTCF-AID and PT gastruloids. +IAA treatment is indicated in red. +Chir stimulation is indicated in grey. Samples were collected every 12h for CTCF-AID gastruloids (orange arrows) and every 24h for PT gastruloids (purple arrows). B. Principal component analysis performed in the RNA-seq samples. PT samples are colored in purple and CTCF-AID are in orange. Circles represent the NT gastruloids and triangles the +IAA@48h treatment. C. Alluvial plot of upregulated (red), downregulated (blue) and non-significant (grey) genes between CTCF-AID NT and CTCF-AID +IAA@48h gastruloids through time. D. Clustered heatmap of differentially expressed genes at 60h and their expression dynamics at later timepoints. E. CTCF Chip-seq signal at the transcription start site (TSS) of downregulated genes at 60h. The Chip-seq data was collected on mESC in Nora et al., 2017 . F. Schematic of CTCF sites considered in follow up analysis (G and H). CTCF binding sites needed to have a CTCF motif and a CTCF peak on Chip-seq. G. Fractions of transcription start sites (TSS) of differentially expressed genes (TSS category) at different timepoints of gastruloid development that have CTCF binding within 1kb. H. Fraction of TSS with CTCF binding within 1kb (G) where the CTCF motif has the same orientation as transcription.

Article Snippet: The full CTCF coding sequence (1-735 amino acids) was amplified by PCR using pKS004-pCAGGS-3XFLAG-CTCF-eGFPk was a gift from Elphege Nora (addgene plasmid #156438) as template.

Techniques: Gene Expression, RNA Sequencing, Expressing, ChIP-sequencing, Binding Assay