Draq7 dye

For the recycling assay, the infection, blocking, and primary staining steps were similar to the pulse-chase assay, except that the cells were stained for 20 min at 37°C.
C with 5% CO₂. After staining, acid-stripping was performed by placing the samples on ice, washing for 1 min with ice-cold DPBS, and stripping for 1 min with chilled acid-stripping buffer. The cells were then washed 2 × 1 min with ice-cold DPBS, reblocked for 3 min in complete RPMI media supplemented with 10% BSA, and then stained with Alexa Fluor 488-labeled mouse anti-human kappa light chain secondary antibody (no. MH10520; Invitrogen) at 1:25 dilution for 1 h at 37°C with 5% CO₂. For the assays utilizing DRAQ7, the DRAQ7 dye (no. D15105; Invitrogen) was included in the secondary stain at 1:100 dilution. The samples were then fixed with 4% paraformaldehyde and 0.2% glutaraldehyde and mounted on slides identical to the pulse-chase assay.

For homeostasis 3–5 days old Ore R female flies were fed standard lab food at 29°C for 6 days. For recovery d2 3–5 days old Ore R female flies were fed 2%DSS for 4 days at 29°C and then transferred to standard food at 29°C for 2 days. Single-Nucleus suspension and FACS: Single-nucleus suspension was conducted as described by Li et al^{127 (link)}. Briefly, ~70 guts per condition were dissected in cold Schneider’s medium, flash-frozen and stored at −80°C. Prior to FACs sorting, samples were spined down and Schneider’s medium was exchanged with homogenization buffer [250mM Sucrose, 10mM Tris pH8, 25mM KCl, 5mM MgCl, 0.1% Triton-X, 0.5% RNasin Plus (Promega, N2615), 50x protease inhibitor (Promega G6521), 0.1mM DTT]. Using 1ml dounce (Wheaton 357538), nuclei were released by 20 loose pestle strokes and 40 tight pestle strokes while keeping samples on ice and avoiding foam. Next, nuclei were filtered through 5 ml cell strainer (40 μm), and using 40 μm Flowmi (BelArt, H13680-0040). Nuclei were centrifuged, resuspended in PBS/0.5%BSA with 0.5% RNase inhibitor, filtered again with 40 μm Flowmi and stained with DRAQ7^™ Dye (Invitrogen, D15106). Single nuclei were sorted with Sony SH800Z Cell Sorter at PCMM Flow Cytometry Facility at Harvard Medical School and 100k nuclei per sample were collected in PBS/BSA buffer.