Chromium next gem chip g single cell kit

scRNAseq using the CITE-seq method (19 (link)) was essentially carried out as described in Abidi et al. (20 (link)). For each experiment, cells were marked with viability dye (Fixable Viability Dye eFluor 450, 1/1000 in PBS without azide or protein, Invitrogen) for 25 min. Living cells were sorted on an ARIA III (BD Biosciences) and marked with conjugated DNA sequences (HashTag Oligonucleotide, HTO, Chromium Single Cell 3’ Feature Barcode Kit, PN-1000079) specific of the donor and the experimental condition following CITE-seq protocols (19 (link)). Cells were then pooled with similar amounts and 20 000 total cells were loaded onto a Chromium controller (10X genomics) (Chromium Next GEM Single Cell 3’ Kit v3.1, ref PN-1000121; Chromium Next GEM Chip G Single Cell Kit, ref PN-1000120). Libraries were then prepared and sequenced on a Nova-Seq 6000 (Illumina) at the GenoBird platform (IRS-UN, CHU Nantes). Raw reads were analyzed using FastQC for quality controls and were then processed using CellRanger pipeline (v3.1.0) with default parameters). Generated FASTQ files were aligned to the reference genome GRCh38.

The utilization of microdroplets in conjunction with barcoded primer beads enabled the capture of individual cells through the implementation of a droplet-based ultra-high throughput system for parallel gene expression detection. The Chromium Next GEM Single Cell 3ʹ Kit v3.1, developed by 10x Genomics (Part Number 1000268), was employed in the creation of gene expression (GEX) libraries. The generation of gel bead-in-emulsions, as well as the reverse transcription (RT) of the single-cell suspensions, was accomplished through the utilization of the 10x Genomics Single cell chip (Chromium Next GEM Chip G Single Cell Kit, Part Number 1000120, and an additional Dual Index Kit TT Set A, Part Number 1000215) run on the Chromium Controller (Part Number 110211) developed by 10x Genomics. The generated cDNA was amplified in order to produce GEX libraries after the RT step, subsequently quantified through the use of Qubit 3.0 fluorometer (Life Technologies, Part Number 15387293) and assessed through the utilization of HS DNA chips (Agilent Technologies, Part Number 5067-4627) in combination with the 2100 Bioanalyzer (Agilent Technologies, Part Number G2939BA). The Novaseq 6000, developed by Illumina, was employed for massively parallel sequencing.

VSMCs were oligo‐tagged with antimouse antibodies (TotalSeq A, Biolegend). The cell suspension was loaded on a 10x Genomics Chromium instrument. The libraries were prepared using Chromium Single Cell 3′ Library & Gel Bead Kit v3.1, PN‐1000268, the Chromium Next GEM Chip G Single Cell Kit PN‐ 1000120 and the Dual Index Kit TT, Set A PN‐1000215, (10x Genomics). Amplified cDNA was evaluated on an Agilent BioAnalyzer 2100 using a High Sensitivity DNA Kit (Agilent Technologies) and final libraries on an Agilent TapeStation 4200 using High Sensitivity D1000 ScreenTape (Agilent Technologies). Individual libraries were diluted to 2 nM and pooled for sequencing. Pools were sequenced with 100 cycle run kits (28 bp Read1, 8 bp Index1, and 91 bp Read2) on the NovaSeq 6000 Sequencing System (Illumina). Seurat package was used for quality control, data filtering, dimensionality reduction, differential gene expression analysis, and uniform manifold approximation.

Using a Chromium Next GEM Single Cell 3’ Kit v3.1, 4 rxns (1000269, 10X Genomics), Chromium Next GEM Single Cell 3ʹ Gel Bead Kit v3.1, 16 rxns (PN-1000122) and Chromium Next GEM Chip G Single Cell Kit (PN-1000120, 10X Genomics), the peritoneal cells suspensions in PBS, at 2000 cells per ul, 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 were added to a chip to create GEMs. Cells were lysed and the bead captured poly(A) RNA was barcoded during reverse transcription in Thermo Fisher 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 was conducted using Agilent Genomic DNA ScreenTape Analysis kit (5067–5366 for Genomic DNA Reagents and 5067–5365 for Genomic DNA ScreenTape) in the TapeStation system. scRNA-seq libraries were constructed using a Chromium Next GEM Single Cell 3ʹ Library Kit v3.1 (PN-1000158, 10X Genomics) and Single Index Kit T Set A, 96 rxns (PN-1000213, 10X Genomics). 10x Genomics Single Cell Gene Expression libraries were sequenced on a NextSeq 2000 run. The sequencing run was setup as a 28 cycles + 90 cycles non-symmetric run. Demultiplexing was done allowing 1 mismatch in the barcodes.