GSM9288587: Peri-implantitis_3; Homo sapiens; RNA-Seq
Split tissues were placed immediately in ice-cold, calcium-free and magnesium-free 1× PBS for overnight shaking and incubation with 1:1,000 ROCK inhibitor (Sigma-Aldrich, Y27632). Cold protease dissociation cocktail was made using cold 1× PBS, 2× DNase (2.5mg ml−1 , Sigma-Aldrich, 9003-98-9) and 1:1,000 ROCK inhibitor. Protease (Sigma-Aldrich, P5380) was then added to a concentration of 100mg ml−1 . Sterilized dissecting forceps (Roboz) were used to mechanically separate gingival into several pieces. The enzymatic cocktail was incubated with gingival tissues for 35min, but progress was checked every 10min with 10-μl aliquots. Dissociation was completed when >75% of the solution was single cells, and then gingival single cells were pelleted at 1,800 relative centrifugal force and washed twice with an appreciable volume of a cocktail of cold culture medium, 1:1,000 Y-27632 and 10% FBS. After dissociation, viability was checked using Trypan blue (viability was confirmed >80%), and red blood cell (RBC) lysis was achieved using a 1× RBC buffer (BioLegend, 420301). A fluorescence-activated cell sorting (FACS) buffer (a mixture of PBS, 1% FBS and 1:1,000 ROCK inhibitor) was used throughout the resuspension and cell sorting process. Single cells were enriched using a 35-mm Falcon cell strainer (Thermo Fisher Scientific). Before FACS, the cell pellet was resuspended in the FACS buffer and stored on ice. To isolate living cells by FACS, rat anti-CD49f Alexa Fluor 647 (a6-Integrin, BioLegend clone GoH3) was added to cells and incubated for 60min on ice. Sytox (Thermo Fisher Scientific, 1:1,000) was added 5min before FACS to evaluate live/dead cells using a Sony SH800S Cell Sorter, with the following controls: anti-CD49f Alexa Fluor 647-negative and Sytox Dead Cell Stain-negative cell suspensions. Cells were sorted ~6 counts per s, and live cells were sorted into tubes directly for 10x capture, library preparation and scRNA-seq. Gel beads in emulsion (GEMs) were broken, and the complementary DNA (cDNA) libraries were purified using Dynabeads MyOne SILANE (Invitrogen) before 12 amplification cycles. Amplified libraries were purified with SPRIselect magnetic beads (Beckman Coulter) and quantified using an Agilent Bioanalyzer High Sensitivity DNA chip (Agilent Technologies). Fragmentation, end repair, A-tailing and double-sided size selection using SPRIselect beads were then performed. Illumina-compatible adapters were ligated onto the size-selected cDNA fragments. Adapter-ligated cDNA was then purified using SPRIselect beads. Uniquely identifiable indexes were added during 12 amplification cycles. The finalized sequencing libraries were then purified using SPRIselect beads, visualized using the Bioanalyzer High Sensitivity DNA chip, quantified with the KAPA SYBR FAST Universal qPCR Kit for Illumina (Roche) and StepOnePlus Real-Time PCR System (Applied Biosystems), normalized to 4nM and pooled. Pooled libraries were sequenced on a NextSeq 500 machine (Illumina). Libraries were denatured and diluted following standard Illumina protocol, spiked with 1% PhiX sequencing control (Illumina), loaded onto the flow cell at 1.8 pM and sequenced in paired-end format (Read 1: 28bp, Read 2: 91bp) to a total depth of 523million read pairs passing quality filters.