Chromium single cell 5 library gel bead kit

Chromium Single Cell 5′ Library & Gel Bead kit, Chromium Single Cell 5′ Library construction kit, Chromium Single Cell A Chip Kit, and Chromium i7 Multiplex kit (10X Genomics) were used for scRNAseq of CSF cells. We followed 10x Genomic’s User Guide for library construction. The only change we made to their protocol was in Step 1, GEM Generation & Barcoding. The user guide recommends loading a certain volume of cell suspension stock depending on the concentration of the cell suspension and the user’s desired cell recovery. However, because CSF contains such low cell numbers, we loaded all the cells that were resuspended in 10 μl of sorting buffer. We then added the 10 μl of cell suspension and 21.7 μl of nuclease free water, which results in the same total volume of cell suspension/water that the user guide recommends. After library construction, libraries were sequenced by Novogene on a Novoseq S4 sequencer and FASTQ files were generated by Novogene. Cell Ranger v.3.0.2 was used to generate gene-expression matrices for CSF cells. Reads from the 10X v.2 5′ paired library were mapped to the human genome build GRCh38 3.0.0. The 5′ gene-expression libraries were then analyzed with the Cell Ranger count pipeline and the resulting expression matrix was used for further analysis in the Seurat package v.3.0.

Encapsulation of lymphocytes and DNA-barcoded gel beads was performed using the Chromium controller (10x Genomics, PN-110203). Briefly, 1.4x10⁴ to 1.7x10⁴ cells (in reverse transcription mix) were loaded per channel for a targeted recovery of 8x10³ to 1x10⁴ cells per sample. Reverse transcription and preparation of single-cell transcriptome, BCR and TCR libraries was performed according to the manufacturer’s instructions (CG000086 manual, RevM, 10x Genomics) and using the following kits: Chromium Single Cell 5’ Library & Gel Bead Kit (PN-1000006), Chromium Single Cell 5’ Library Construction Kit (PN-1000020), Chromium Single Cell V(D)J Enrichment Kit, Human T Cell (PN-1000005), Chromium Single Cell V(D)J Enrichment Kit, Human B Cell (PN-1000016), Chromium Single Cell A Chip Kit (PN-1000009), Chromium i7 Multiplex Kit (PN-120262).

For scRNAseq, libraries were generated using Chromium Single Cell 5′ Library & Gel Bead kit (1000006; 10× Genomics) from 2.5 × 10⁴ viable CD45⁺ cells sorted from 4T1 tumors.

Single-cell suspensions were loaded onto a Chromium Single Cell Chip (10x Genomics) according to the manufacturer’s instructions for coencapsulation with barcoded gel beads at a target capture rate of approximately 10,000 individual cells per sample. Captured mRNA was barcoded during cDNA synthesis, and the barcoded cDNA was converted into pooled single-cell RNA-seq libraries for Illumina sequencing by using the Chromium Single Cell 5′ Library & Gel Bead Kit (10x Genomics) according to the manufacturer’s instructions. All samples were processed simultaneously with the Chromium Controller (10x Genomics) and the resulting libraries were prepared in parallel in a single batch. All libraries were sequenced with a NovaSeq 6000 sequencing system. Demultiplexing of sequencing results, barcode processing, read alignment, and unique molecular identifier (UMI) counting were performed using the 10x Cell Ranger analysis pipeline v6.0. Further quality control, feature selection, dimension reduction, unsupervised clustering, and differential expression analyses were performed using the Seurat R package v4.1.1 (53 (link)). Data are available at the Zenodo database (https://doi.org/10.5281/zenodo.5544128).