ENDOMETRIAL POLYPS

Cell capture and cDNA synthesis Using single cell 3' Library and Gel Bead Kit V3 (10x Genomics, 1000075) and Chromium Single Cell B Chip Kit (10x Genomics, 1000074), the cell suspension (300-600 living cells per microliter determined by Count Star) was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. In short, single cells were suspended in PBS containing 0.04% BSA. About 6,000 cells were added to each channel, and the target cell will be recovered and estimated to be about 3,000 cells. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53°C for 45 min, followed by 85°C for 5 min, and hold at 4°C. The cDNA was generated and then amplified, and quality assessed using an Agilent 4200 (performed by CapitalBio Technology, Beijing). According to the manufacture’s introduction, Single-cell RNA-seq libraries were constructed using Single Cell 3’ Library and Gel Bead Kit V3. The libraries were finally sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with pair-end 150 bp (PE150) reading strategy (performed by CapitalBio Technology, Beijing)

本实验采用10x Genomics公司的Single Cell 3' Library and Gel Bead Kit V3（货号：1000075）与Chromium Single Cell B Chip Kit（货号：1000074）完成细胞捕获与cDNA合成。将经Count Star细胞计数仪测定、活细胞浓度调整为每微升300~600个的细胞悬液，加载至10x Genomics的Chromium单细胞控制器中，依照厂商官方规程在油包水体系中生成包裹单细胞的凝胶微珠乳液（Gel Bead in Emulsion，简称GEMs）。简言之，将单细胞重悬于含0.04%牛血清白蛋白（BSA）的磷酸盐缓冲液（PBS）中，每个反应通道加入约6000个细胞，最终预计可回收目标细胞约3000个。捕获的细胞经裂解后，释放的RNA在独立的GEMs中通过逆转录反应带上特异性条码。逆转录反应在S1000™ Touch型触控梯度PCR仪（Bio-Rad）中进行：53℃反应45分钟，随后85℃灭活5分钟，最后于4℃保温。合成得到的cDNA经扩增后，采用安捷伦4200生物分析仪完成质量质控，该检测步骤由北京博奥生物科技（CapitalBio Technology）完成。依照试剂盒厂商的操作指南，采用Single Cell 3' Library and Gel Bead Kit V3构建单细胞RNA测序文库。最终采用Illumina NovaSeq6000测序仪对构建完成的文库进行测序，测序策略为双端150bp（PE150），每个细胞的测序深度不低于100000条reads，该测序步骤同样由北京博奥生物科技（CapitalBio Technology）完成。