Fig. 1. NICHE-seq accurately and reproducibly depicts the cellular composition of defined niches.

(A) TPLSM images of naive inguinal lymph nodes (LNs) from PA-GFP host mice, before and after photoactivation of subregions (green). Adoptively transferred TdTomato⁺ cells (red) and CFP⁺ B cells (cyan) mark the T cell area and the B follicles, respectively. Second harmonic generation was used to detect collagen fibers (cyan). (B) Cell type distribution in the unlabeled LN and photoactivated B cell follicles and Tcell areas, measured by flow cytometry. Error bars represent standard error from three independent experiments. (C) Gene expression profiles of 3900 single cells from photoactivated B cell follicles or Tcell areas of naive inguinal LNs, grouped into five clusters (table S1). The color bar at the top indicates each cell’s origin. Expression is normalized by total cell count and highest gene value. (D) Relative enrichment of different cell types in each subregion (log₂ fold change compared with the total naive LN). Error bars represent 90% confidence intervals. *q < 0.001; Fisher test. (E) Same as (C), but for 8100 single cells from inguinal LNs, 72 hours after infection with LCMV, grouped into seven clusters (table S2). Mono, monocytes; act, activated; inf, inflammatory. (F) Same as (D), but for LCMV-infected cells.