Extended Data Fig. 1: Generating a WAPL-AID degron line.
From: WAPL maintains a cohesin loading cycle to preserve cell-type-specific distal gene regulation
a, Homologous recombination strategy for tagging the endogenous Wapl gene with AID-eGFP. Note that the targeting construct contains a NeoR/KanR resistance gene, but that this has not been used for selecting the first clone. Middle panel shows PCR validation (primers highlighted in left panel) showing homozygous integration of the donor vector (n=1). Right panel shows Sanger sequencing results confirming in-frame tagging of AID tag. b, Left panel shows live cell imaging of WAPL in untreated (0h) and treated (24h) cells (representative images from 3 separate fields). DNA is visualized with Hoechst. Right panel show immunostaining of cohesin subunit RAD21 (representative images from 100 independent cells). DNA is labeled with DAPI. c, WAPL-AID-GFP levels were measured by FACS (gated on all cells besides debris). High temporal resolution shows rapid depletion of WAPL-AID-GFP upon IAA treatment. Median GFP intensity is quantified in bottom plots. Signals are the average of three experiments. Dots indicate mean values. Error bars indicate standard deviation. d, ChIP-seq of WAPL and CTCF before and after IAA treatment. Heatmaps show the distribution of signal for the peaks called in the untreated cells. Average signal is shown above the heatmap. ChIP-seq data is calibrated using spiked-in human HEK-293T cells. Middle two heatmaps show WAPL ChIP-seq signal derived from the human cells.
