Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Bone chips (endosteum) were harvested by crushing bones and washing away marrow with ice-cold HBSS. Marrow plugs (central marrow) were harvested by flushing intact plugs with ice-cold HBSS. All samples were subjected to collagenase digestion as indicated in the manuscript to liberate stromal cells, filtered through 100 micron mesh, and stained for surface protein expression. Live Ter119-/CD45- cells from each compartment were directly sorted into filter-sterilized alphaMEM supplemented with 10% FBS and purity was confirmed to be >95% by running freshly sorted cells on the same sorter. Libraries were constructed following manufacturer's instructions for Chromium Single Cell 3' Library & Bead Kit v2 (10X Genomics). 5,000 cells were loaded for each sample and samples were sequenced in a HiSeq4000 sequencer (Illumina) to obtain an average of ~286 million reads per sample