Chromium single cell b chip kit

Using a Chromium Single Cell 3ʹ GEM Kit v3 (10x Genomics, 1000094), Chromium Single Cell 3ʹ Gel Bead Kit v3 (10x Genomics, 1000093) and the Chromium Chip B Single Cell Kit (10x Genomics, 1000153), the anti-IgM Fab′2 activated splenic B cell suspensions in RPMI plus 2.0% FBS, at 1,000 cells per µl, were loaded onto a chromium single-cell controller (10x Genomics) to generate single-cell gel beads-in-emulsion (GEMs) according to the manufacturer’s protocol. Briefly, approximately 10,000 cells per sample (n = 2) were added to chip B to create GEMs. Cells were lysed, and the bead captured poly(A) RNA was barcoded during reverse transcription in Thermo Fisher Scientific Veriti 96-well thermal cycler at 53 °C for 45 min, followed by 85 °C for 5 min. cDNA was generated and amplified. Quality control and quantification of the cDNA were conducted using Agilent’s High Sensitivity DNA Kit (5067-4626) in the 2100 Bioanalyzer. scRNA-seq libraries were constructed using a Chromium Single Cell 3ʹ Library Kit v3 (10x Genomics, 1000095) and indexed with Chromium i7 Multiplex Kit (10x Genomics, 220103). The libraries were sequenced using an Illumina HiSeq2500 sequencer with a paired-end, single-indexing strategy consisting of 28 cycles for read 1 and 91 cycles for read 2. Multiple sequencing runs were performed to achieve greater sequencing depth for each barcode.

Based on the previous studies,²⁰ (link) four typical synovial tissues (two AF group patients and two control group patients) were mechanically minced and enzymatically digested with Liberase TL (100 g/mL; Roche) and DNAse I (100 g/mL; Roche) for 30 min at 37°C. Red Blood Cell Lysis solution was used to lyse erythrocytes after fetal calf serum was used to stop the digestion process (Milteny Biotec). The LUNA automated cell counter was used to wash and count the cells (Logos Biosystems). Using the Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3, the Chromium Chip B Single Cell Kit (10 Genomics), and the Chromium controller (all 10 Genomics), a total of 15 000 unsorted synovial cells per patient were prepared for single cell analysis. On the Illumina NovaSeq instrument, libraries were sequenced to a depth of 20 000–70 000 reads per cell. The reads were demultiplexed, and aligned to the Ensembl reference build GRCh38.p13, and the unique molecular identifiers were collapsed using CellRanger (V.2.0.2) from 10X Genomics.

Libraries for Single cell RNA seq were generated using Chromium single cell 3′ library & Gel Bead Kit v3 (PN-1000092, 10X Genomics), Chromium Single Cell B Chip kit (PN-1000074, 10X Genomics). Briefly, cells were mixed with reverse transcription (RT) reaction reagent and loaded onto the B chip aiming for 6000 captured cells per a channel. After RT reaction, gel bead-in-emulsions were transferred to tubes and performed RT reaction using thermal cycler (C1000 Touch, Bio-Rad, RRID:SCR_019688). cDNA was purified using Dynabead MyOne SILANE (Thermofisher) and further amplified. Subsequent steps for library construction were performed by the manufacturer’s instruction provided. Quality of the amplified cDNA and final libraries were monitored by Bioanalyzer (Agilent, RRID:SCR_019715). Libraries were sequenced with a 2 × 100 bp paired-end protocol on a Novaseq S4 platform from Illumina to generate minimum 20,000 read pairs per cell.

Single cell suspensions (300–500 living cells per micro liter) were loaded on a Chromium Single Cell Controller (10x Genomics) to generate single-cell Gel Bead-In-Emulsions (GEM) by using single cell 3 ’Library and Gel Bead Kit V3 (10x Genomics, 1000075) and Chromium Single Cell B Chip Kit (10x Genomics, 1000074). 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 was estimated to be about 3,000 cells. Captured cells were lysed and the released RNA was bar-coded 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). Single-cell RNA sequencing 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).

The detailed methods of WGBS and scRNA-seq are described in Supplementary material. In brief, for WGBS, an Acegen Bisulfite-Seq Library Prep Kit (Acegen, Cat. no. AG0311) was applied for library construction. An Illumina HiSeq X Ten platform was used for final sequencing. For scRNA-seq, single cells were isolated from D3 organoids and captured in nanoliter droplets using a Chromium Single Cell B Chip Kit (10x Genomics, 1000074). scRNA-seq libraries were constructed using a Single Cell 3′ Library and Gel Bead Kit V3 (10x Genomics, 1000075), and sequencing was accomplished using an Illumina NovaSeq 6000 sequencer.

Nuclei were loaded onto a Chromium single-cell controller (10x Genomics) to generate single-nucleus gel beads in the emulsion (GEM) using a single-cell 3' Library and Gel Bead Kit V3.1 (10x Genomics, 1000075) and Chromium Single Cell B Chip Kit (10x Genomics, 1000074) according to the manufacturer’s instructions. Approximately 8040 nuclei were captured from each sample. The captured nucleus was lysed, and the released mRNA was barcoded through reverse transcription in individual GEMs. Reverse transcription was performed to generate cDNA using an S1000TM Touch Thermal Cycler (Bio-Rad) with the following parameters: 53°C for 45 min, 85°C for 5 min, and 4°C until further use. The cDNA was then amplified, and the quality was assessed using an Agilent 4200 (CapitalBio Technology).

Tissue samples were processed immediately after the surgical resection. Single-cell suspensions were obtained by mechanical and enzymatic dissociation. By following the manufacturer's protocol, we used the Single Cell 3' Library and Gel Bead Kit V3.1 (10x Genomics, 1000075) and Chromium Single Cell B Chip Kit (10x Genomics, 1000074) to prepare barcoded scRNA-seq. Paired-end 150 bp reads were then generated by sequencing the libraries on the Illumina NovaSeq6000 platform (performed by CapitalBio Technology, Beijing).