Convergent transcription is detected at Igh switch regions in early G1 cells. (A) Experimental layout of the double-sorting strategy to isolate live early G1 cells. After 6-h nocodazole treatment, the Fucci-EGFP⁺ B cells (mostly M phase) were sorted and replated in nocodazole-free medium for 1.5 h. After the second sort, EGFP⁻ small cells (mostly early G1) were purified (see Materials and methods). (B) FACS analysis of the double-sorting strategy. At each step, a small aliquot of cells was fixed and stained with DAPI (quality control, #1–#5). G1, G2/M, and late M populations (including cells in cytokinesis) are indicated on the representative plots. (C) GRO-Seq and convergent transcription (ConvT) profiles at the Igh locus in early G1 cells. Sense (light blue) and antisense transcription (dark blue) are displayed. ConvT regions are shown as green bars, and the numbers indicate the levels of ConvT, which were calculated by the geometric means of sense and antisense transcription reads as described previously (Meng et al., 2014). The arrow shows the direction of sense transcription. Two independent experiments were performed.