ZFP36L2 orchestrates stress-adaptive plasticity in intestinal regeneration and colorectal cancer metastasis (PDO scRNAseq)

Organoids expressing a doxycycline-inducible shRNA targeting ZFP36L2 (shZFP36L2) or a non-targeting control (shCtrl) were cultured with 2 μg/mL doxycycline and subjected to three conditions: (1) HISC medium for 7 days (baseline ISC state); (2) HISC for 7 days followed by intestinal growth factor-free (IGFF) medium for 7 days (differentiation); and (3) HISC for 7 days, IGFF for 7 days, then HISC for 7 days (dedifferentiation).

Organoids were dissociated into single cells and multiplexed using TotalSeq hashtag antibodies (BioLegend). Viable (DAPI-negative) cells were FACS-sorted and pooled in equal numbers across conditions for library preparation using the Chromium Single Cell 3′ v3.1 platform (10x Genomics), targeting up to 10,000 cells per sample. Libraries were sequenced on an Illumina NovaSeq S4 (Read 1: 28 cycles; i7 index: 8 cycles; Read 2: 90 cycles).