Isolation and Sequencing of Periodontal Cells

Dissociated periodontal cells harvested from P25 Col1(2.3kb)-GFP;PTHrP-creER;R26R-tdTomato molars were pooled. Cell sorting was performed using a six-laser Sony Synergy SY3200 (Ex.350/405/488/561/594/685nm) high-speed cell sorter with a 100-μm nozzle. Col1a1(2.3kb)-GFP⁺ cells including tdTomato⁺ cells were directly sorted into ice-cold DPBS/1% FBS, pelleted by centrifugation and resuspended in 30 μl DPBS/1% FBS using wide-bore pipettes. Cell numbers were quantified by Countless II automated Cell Counter (ThermoFisher) before loading onto the Chromium Single Cell 3’ microfluidics chip (10× Genomics Inc., Pleasanton, CA). cDNA libraries were sequenced by Illumina HiSeq 4,000 using two lanes and 50 cycle paired-end read, generating a total of ~ 770 million reads. The sequencing data was first pre-processed using the 10× Genomics Cell Ranger software. For alignment purposes, we generated and used a custom genome fasta and index file by including the sequences of mCherry to the mouse genome (mm10). The scRNA-seq dataset presented herein have been deposited in the National Center for Biotechnology Information (NCBI)’s Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE120108 and GSE168450. The dataset GSE120108 has been published previously (2 (link)).