GSM6290648: Gingiva_hh; Homo sapiens; RNA-Seq
For v_i group, the gingival tissues from interdental space of periodontitis affected site were harvested during guided tissue regeneration procedure. For h_h and v_h group, gingival tissues from interdental space along the extended crevicular incision were harvested during open flap surgery of adjacent affected teeth.Gingival samples were minced into small pieces of less than 1 mm3 by surgical scissors and dissociated into single cells in dissociation solution (2 mg/ml Ⅳ collagenase , 2 units/ml Dispase Ⅱ in Ca2+- and Mg2+-free HBSS) at 37°C for 60 min; 0.1 mg/mL DNase I was added in the last 10 min. Cell suspension was filtered by 70μm cell strainers. Cells were subjected to red blood cell lysis for 10 min and centrifuged and re-suspended (500 g, 10min) twice. After resuspension in defined volumes of PBS + 0.04% BSA, cells were stained with 0.4% Trypan blue, and cell viability was evaluated under microscope. When the viability of cells was higher than 80%, the experiment of library construction was performed. The single-cell library was constructed using the ChromiumTM Controller and ChromiumTM Single Cell 3' Reagent Version 3 Kit (10x Genomics, Pleasanton, CA). Briefly, single cells, reagents and Gel Beads containing barcoded oligonucleotides were encapsulated into nanoliter-sized GEMs (Gel Bead in emulsion) using the GemCode Technology. Lysis and barcoded reverse transcription of polyadenylated mRNA from single cells were performed inside each GEM. The post RT-GEMs were cleaned up by silane magnetic beads, and cDNA were amplified by PCR. cDNA was fragmented and fragments end were repaired, as well A-tailing was added to the 3' end. Solid Phase Reversible Immobilization (SPRI) beads were used to remove large and small fragments. Another double sided SPRI selection was carried out after sample index PCR. The final library quantification was performed by checking the distribution of the fragments size using the Agilent 2100 bioanalyzer, and by using real-time quantitative PCR (QPCR) (TaqMan Probe). The final products were sequenced using the Illumina Hiseq 4000 (BGI-Shenzhen, China).