NextSeq 550 paired end sequencing; Haematopoietic stem and progenitor cells are present in healthy gingiva tissue
Haematopoietic stem and progenitor cells are present in healthy gingiva tissue
All mice were housed under specific-pathogen free conditions and euthanized by approved schedule 1 methods following local guidelines. Single cell suspensions were generated from tissues and incubated with antibody cocktails in FACS buffer (PBS with 2% FCS and 2 mM EDTA) for 15 min at 4°C, then specific populations of interest obtained by sorting cells on a BD Aria cell sorter. Dead cells were excluded by use of a Live/Dead Fixable dye (Biolegend). HSPCs were identified as follows: LSK+MyP: CD45+Lin (CD3ε, TCR-β, CD19, B220, NK1.1, Ter119, Siglec F, Ly6G, FcεR1α, CD11b, CD11c, Ly6C)-Sca-1+/-cKit+.  Gene expression libraries were prepared from single cells using the Chromium Controller and Single Cell 3ʹ Reagent Kits v3 (10x Genomics, Inc. Pleasanton, USA) according to the manufacturer's protocol. Gene expression libraries were prepared from single cells using the Chromium Controller and Single Cell 3ʹ Reagent Kits v3 (10x Genomics, Inc. Pleasanton, USA) according to the manufacturer's protocol. Briefly, nanoliter-scale Gel Beads-in-emulsion (GEMs) were generated by combining barcoded Gel Beads, a master mix containing cells, and partitioning oil onto a chromium chip. Cells were delivered at a limiting dilution, such that the majority (90-99%) of generated GEMs contain no cell, while the remainder largely contain a single cell. The Gel Beads were then dissolved, primers released, and any co-partitioned cells lysed. Primers containing an Illumina TruSeq Read 1 sequencing primer, a 16- nucleotide 10x Barcode, a 12-nucleotide unique molecular identifier (UMI) and a 30-nucleotide poly(dT) sequence were then mixed with the cell lysate and a master mix containing reverse transcription (RT) reagents. Incubation of the GEMs then yielded barcoded cDNA from poly-adenylated mRNA. Following incubation, GEMs were broken and pooled fractions recovered. First-strand cDNA was then purified from the post GEM-RT reaction mixture using silane magnetic beads and amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were then used to optimize the cDNA amplicon size. Illumina P5 & P7 sequences, a sample index, and TruSeq Read 2 sequence were added via end repair, A-tailing, adaptor ligation, and PCR to yield final Illumina-compatible sequencing libraries.