GSM8970691: Citeseq, P8, ADT; Homo sapiens; OTHER
Preparation of single cell suspensions for CITE-seq and spectral flow cytometry was carried out as previously described (Greenwell-Wild et al., 2021). Briefly, tissue fragments were minced and digested using collagenase II (Worthington Biochemical Corporation) and DNase (Sigma) and dissociated with the gentleMACS Dissociator (Miltenyi). Cells were then passed through a 70mm filter (Falcon, Corning), washed, and counted (Cellometer Auto 2000 - Nexcelom). Single cell preparations of between 5x104 and 5x105 single cells from periodontitis samples were stained individually with TotalSeq™-A Human Universal Cocktail, V1.0 (Biolegend, CA) as per the manufacturer's protocol (Suppl. Table 4). 1.7x104 stained cells were then loaded with the reverse transcription mix on the Chromium chip G and partitioned into single cells in Gel Beads-in-emulsion (GEMs) using the 10X Genomics Chromium Next GEM Single Cell 3' Kit v3.1 as per manufacturer's instructions (10X Genomics, CA). cDNA amplification and ADT libraries were prepared as per instructions from Biolegend while gene expression libraries were made as per the 10X Genomics protocol. All amplification steps were performed in an Applied Biosystems Ins (ABI) Veriti 96-well thermal cycler. Quality and quantity of the libraries were assessed using TapeStation (Agilent, CA) and a Qubit fluorometer (ThermoFisher). Libraries were pooled at a concentration of 2nM and sequenced on an Illumina platform (NextSeq 2000, Illumina, San Diego, CA) using the following read lengths: Read 1: 28 base pairs, Index 1: 10 base pairs, Index 2: 10 base pairs, Read 2: 90 base pairs.