Chromium single cell 5 v2 reagent kit

Gel Bead and Multiplex Kit, Chip Kit (10X Genomics) and Chromium Single Cell 5′ Library (10X Genomics, Genomics chromium platform Illumina NovaSeq 6000) were used to convert the harvested single-cell suspensions into barcoded scRNA-seq libraries. Single-cell RNA libraries was made up of the Chromium Single Cell 5′ v2 Reagent Kit (120237; 10X Genomics) according to the manufacturer’s instructions, and FastQC software was used to inspect library quality. All sequenced data were preliminarily processed by CellRanger software (version 4.0; 10X Genomics). The count pipeline of the CellRanger software Suite was applied to demultiplexed and barcoded sequences. Seurat package (version 3.0) was applied to filtration, normalization, dimensionality reduction, cell clustering, and differential gene expression analysis of the processed data, on the foundation of the calculated single-cell expression matrix by CellRanger. Cells with less than 200 genes detected and a mitochondrial gene ratio of more than 20% were excluded. A total of 53,701 cells (Blank, 15,617 cells; Model, 38,084 cells) were analyzed after quality control.

For single-cell RNA sequencing, single-cell suspensions were prepared from half-spleens of NP-OVA immunized SellCreERT2 ROSAtdT mice on days 7 and 21 after immunization. Samples were indexed with TotalSeqC (BioLegend) cell surface antibodies, and CD4⁺, CD62^low, CD44^hi, PD1^hi, CXCR5^high, tdTomato⁺ Tfh cells were purified by flow cytometry, pooled, and loaded onto a Chromium Controller (10x Genomics). Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 5′ v2 Reagent Kit (10× Genomics) according to the manufacturer’s protocol. Libraries were loaded onto an Illumina NextSeq with the mid-Output Kit (150 paired end) for V-D-J analysis or NOVAseq for single-cell gene expression. Hashtag indexing was used to demultiplex the sequencing data and generate gene-barcode matrices, respectively.

Single-cell suspensions were converted to barcoded scRNA-seq libraries using the Chromium Single Cell 5′ Library (10X Genomics, Genomics chromium platform Illumina NovaSeq 6000), Gel Bead and Multiplex Kit, and Chip Kit (10X Genomics). The Chromium Single Cell 5′ v2 Reagent Kit (120237; 10X Genomics) was used to prepare single-cell RNA libraries according to the manufacturer’s instructions. FastQC software was used to check library quality. Preliminary processing of sequenced data was performed using CellRanger software (version 4.0; 10X Genomics). The count pipeline in the CellRanger software Suite was applied to demultiplex and barcode the sequences. Based on the calculation of the single-cell expression matrix by CellRanger, filtration, normalization, dimensionality reduction, clustering, and differential gene expression analysis were conducted using the Seurat package (version 3.0)⁶⁹ . Before filtration using the Seurat package, we removed the cell population expressing HBB, HBA1, and several light and heavy chain transcripts, which are considered red blood cell-contaminated^{17 (link)}. Next, cells with fewer than 200 genes detected and a mitochondrial gene ratio of greater than 15% were excluded. A total of 96,465 cells (preSU, 45,797 cells; postSU, 50,668 cells) were analyzed after quality control.

LCMV-specific (GP_66-77 tetramer⁺) CD4⁺ T cells were harvested from the spleens of two LCMV Cl13–infected mice on day 10 after infection and were FACS sorted using a FACS-Aria Sorter (BD Biosciences). Sorted cells were loaded onto the 10× Chromium Controller with a target cell number of 5000 per mouse. scRNA-seq libraries were prepared using the Chromium Single Cell 5’ v2 Reagent Kit (10× Genomics) according to the manufacturer’s protocol. Two libraries were then quantified using the KAPA library quantification Kit and then were loaded onto an Illumina NextSeq 500 sequencer with the NextSeq 500/550 High Output Kit v2.5 (150 cycles; 20024907; Illumina) with the following conditions: 26 cycles for read 1, 98 cycles for read 2, and 8 cycles for the i7 index read. Raw sequencing data were downloaded from Illumina BaseSpace, then demultiplexed and converted to gene-barcode matrices using the ‘mkfastq’ and ‘count’ functions in Cell Ranger v3.0 (10× Genomics). A total of 4394 and 3613 cells were recovered from samples M1 and M2, respectively.

Lymphocytes isolated from the small intestine epithelium or LP were isolated as described above and indexed with TotalSeqC Hashtag (BioLegend) cell surface antibodies, with two barcodes used per sample for deeper multiplexing. Total CD4⁺ T cells were sorted, pooled, and immediately loaded onto a Chromium Controller (10× Genomics). For Sell^Tomato sorted Tomato⁺ and Tomato⁻ (CD62L⁻, CD4⁺ T) cells were pooled at a ∼1:3 ratio. 5′ Gene expression, VDJ, and Feature Barcode libraries were prepared using the Chromium Single Cell 5′ v2 Reagent Kit (10× Genomics) according to the manufacturer’s protocol at the Genomics core of The Rockefeller University. Libraries were sequenced on Illumina NextSeq500 or NovaSeq 6000. Hashtag indexing was used to demultiplex the sequencing data and generate gene-barcode matrices.