Chromium single cell a chip kit

Single-cell RNA-seq libraries were constructed using a Single Cell 3′ Library and Gel Bead Kit (v3.1; 10X Genomics, 1000121) and a Chromium Single Cell A Chip Kit (10X Genomics, 1000120) according to the manufacturer’s instructions. Single cells were suspended in PBS containing 0.04% BSA (700–1,200 living cells per ml as determined using the LUNA-FL cell counter) and loaded onto a Chromium single-cell controller (10X Genomics) to generate single-cell gel bead-in-emulsion (GEMs) according to the manufacturer’s protocol. Approximately 10,000 cells were added to each channel and approximately 10,000 target cells were captured from each sample. The captured cells were lysed, and the released RNA was barcoded through reverse transcription in each GEM. Reverse transcription was performed using a C1000 Touch Thermal Cycler (Bio-Rad) at 53°C for 45 min, followed by 85°C for 5 min, and held at 4°C. The resulting cDNA was amplified and then assessed for quality using an Agilent 4200 TapeStation. The libraries were paired-end (150 bp) sequenced using the Illumina NovaSeq 6000 system. The above experiments were carried out by BioMiao Biological Technology Co., Ltd, Beijing, China.

After isolation of human CD271+ BM-MNCs, we applied the Chromium single cell gene expression platform (10x Genomics, Pleasanton, CA, USA) for scRNA-seq experiments. Cell suspensions were loaded on a Chromium Single Cell Controller (10x Genomics) to generate single-cell gel beads in emulsion (GEMs) by using Single Cell 3' Library and Gel Bead Kit V3 (10x Genomics, Cat# 1000092) and Chromium Single Cell A Chip Kit (10x Genomics, Cat#120236) according to the manufacturer's protocol. Briefly, single cells were suspended in 0.04% BSA-PBS. Cells were added to each channel, captured cells were lysed, and the released RNA were barcoded through reverse transcription in individual GEMs²⁷. GEMs were reverse transcribed in a C1000 Touch Thermal Cycler (Bio Rad, Hercules, CA, USA) programmed at 53 °C for 45 min, 85 °C for 5 min, and held at 4 °C. After reverse transcription, single-cell droplets were broken, and the single-strand cDNAs were isolated and cleaned with Cleanup Mix containing DynaBeads (Thermo Fisher Scientific). cDNAs were generated and amplified, and the quality was assessed using the Agilent 4200.

Using single-cell 5′ Library and Gel Bead Kit (10x Genomics, 1000006) and Chromium Single Cell A Chip Kit (10x Genomics, 120236), 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. Then the cells were added to each channel, and the target cell will be recovered. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs [23 (link)]. 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, Beijing).

Libraries for scRNA-seq were generated using Chromium Single Cell 3’ Library & Gel Bead Kit v2 (10X Genomics, PN-120237) and Chromium Single cell A Chip Kit (10X Genomics, PN-120236), and Chromium i7 Multiplex Kit (10X Genomics, PN-120262). All the processes were performed according to the manufacture’s protocol. Briefly, cells were counted and diluted to 4 × 10⁵ ~ 2 × 10⁶/ml concentration. The cell suspension was added to the reverse transcription reaction mixture and loaded onto A Chip aiming for capture of 3000 cells. Gel Bead-In-Emulsions (GEMs) were generated using Chromium Controller. GEMs were incubated in a thermal cycler for reverse transcription. After cDNA recovery from GEMs, cDNA was amplified using a thermal cycler. The size and quantity of amplified cDNA were analyzed using Bioanalyzer (Agilent). Subsequent procedures including enzymatic fragmentation, end repair, A-tailing, adaptor ligation, and sample index PCR for the sequencing library were performed using the purified cDNA. The quality of the final library was analyzed using Bioanalyzer High Sensitivity Chip (Agilent). Libraries were pooled and sequenced through the Hiseq2500 platform (Illumina) with the following parameters: Read1: 26 cycles, i7 index: 8 cycles, i5 index: 0 cycles, and Read2: 98 cycles.