Cerebrospinal fluid regulates skull bone marrow niches via direct access through dural channels

We have previously shown that skull bone marrow derived myeloid cells are different from their blood derived counterparts. Whether or not cues from the CNS microenvironment differentially shape the skull bone marrow niche relative to peripheral bone marrow niches is unknown. To test this, we performed scRNAseq of skull and peripheral bone marrow niches. Tibias and skulls were harvested from WT 8 week old male C57BL/6J mice and the surrounding flesh was removed. For skulls the dura was peeled and removed with fine forceps. Both the tibia and skull were then cut into small pieces using sterile scissors and mechanically dissociated in FACS buffer with a pestle, followed by a filtration step through a 70-μm cell strainer. Samples were centrifuged for 5 minutes at 420 x g, and red blood cell lysis performed with ACK lysis buffer. Samples were washed in fluorescence-activated cell sorting (FACS) buffer (PBS with 2% bovine serum albumin and 1 mM EDTA), stained with DAPI, and Viable (DAPI-) single cells were sorted on a BD FACSAria II (BD Biosciences) then sent for single-cell RNA sequencing.