GSM9206804: Periodontal ligament, immature; Homo sapiens; RNA-Seq
Tissues were digested containing collagenaseⅠ(3mg/ml) and Dispase Ⅱ (4mg/ml) at 37℃. Counted every 10 minutes, the dissociated cells were passed through a 70μm filter, centrifuged at 400g at 4℃ for 5 min. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer (Miltenyi) and incubated on ice for 5 min to lyse red blood cells. After cracking red, the reaction was terminated with PBS+2%FBS. Centrifuged at 400g at 4℃ for 5 min. Then discard the supernatant and re-suspend, centrifuged at 300g at 4℃ for 5 min, discard the supernatant and re-suspend, the dissociated cells were passed through a 40μm filter element, and counted by Countstar automatic counter (Countstar Rigel S5). We loaded around 17000 cells into the 10x Chromium Controller. We used the Single-Cell 3′ Reagent Kit v3.1 according to the manufacturer's protocol. GEM-Reverse Transcription (GEM-RT) was performed in a thermal cycler: 53℃ for 45 min, 85℃ for 5 min, and held at 4℃(P24-P31).After reverse transcription and cell barcoding, emulsions were broken and cDNA was isolated and purified with Cleanup Mix containing DynaBeads and SPRIselect reagent (Thermo Fisher Scientific), followed by PCR amplification. The cDNA was then evaluated using the Agilent Bioanalyzer. For RNA-seq library construction, amplified cDNA was fragmented and end-repaired, double-sided size-selected, PCR-amplified with sample indexing primers, and double-sided size-selected (P32-P38). Libraries prepared according to the manufacturer's user guide were purified and profiled for quality assessment. Single-cell RNA library was sequenced by an Illumina NovaSeq 6000 sequencer with 150 bp paired-end reads (P39-51).