Molecular and cellular characterization of planarian stem cell microenvironments

A Slide-seqV2 atlas of intact and regenerating tail fragments at 6 hours and 48 hours post amputation (hpa). Intact 8 mm animals were removed from recirculation culture and 1.25 mm biopsy punches were used to amputate tail fragments. Regenerating tail fragments were embedded in OCT around an intact 2-3 mm worm, sectioned, and 2 adjacent sections were processed using the Slide-seqV2 protocol (Stickels et al. Nature Biotechnology 2021). Captured mRNA was sequenced on an Illumina platform. RNA-seq of cells positive and negative for matrix metalloproteinase-1 (mmp-1) mRNA. Overall design: For each timepoint 8-9 tail fragments were arrayed around a single intact animal in OCT prior to freezing. Tissue sections were slowly removed from the block until reaching the middle of the worm fragments. Two adjacent sections were then cut and laid onto Slide-seqV2 pucks for mRNA capture and library preparation. Each puck resulted in one library, resulting in 4 total libraries that were pooled and sequenced on 2 NextSeq flow cells. Mmp-1 positive cells were identified by performing HCRv3 on fixed cells dissociated from planarians at three timepoints: intact worms, as well as 6 and 48 hours post amputation. Positive cells were sorted by flow cytometry for library construction and sequencing.