Chromium next gem single cell 3 kit v3

The droplet-based 10X Chromium system employs the GemCode technology, which utilizes gel beads in emulsion to achieve barcoding [46 (link)]. This involves combining a suspension of single cells with gel beads containing barcoded oligonucleotides and reagents for reverse transcriptase (RT) in an oil environment, resulting in the formation of nanoliter-scale droplets that facilitate cDNA synthesis. Subsequently, the droplets are pooled, dissolved, and used to create a sequencing library containing unique molecular identifiers (UMIs). The microfluidic chips used in this system can accommodate up to eight samples simultaneously, each containing up to 10,000 cells. Samples obtained from four PCa patients were converted to scRNA-seq libraries using the Chromium Next GEM Single-Cell 3′Kit v3.1 (10X Genomics) as described previously [35 (link)]. Three out of these four patients (∼48,000 cells) were processed in parallel with the BD Rhapsody platform.

Each cell suspension was subjected to 3′ single-cell RNA sequencing using 10X Chromium Controller with accompanying Chromium Next GEM Single Cell 3’ Kit v.3.1 (10X Genomics, Pleasanton, CA), following the manufacturer’s instructions. Libraries were sequenced on a NextSeq Illumina 5000 platform and mapped to the GRCm38/91 mouse reference genome using the 10x Cell Ranger analysis tool.

Single-cell suspensions were processed using Chromium Next GEM Single Cell 3’ Kit v3.1 (10x Genomics, Pleasanton, CA). Briefly, up to 16,500 cells were resuspended in the reaction mix and loaded into the Chromium Next GEM Chip G, followed by loading the 3ʹ v3.1 Gel Beads and the partitioning oil. Chip G was placed into the 10x Chromium controller for cell partitioning. After partitioning, 100µL of GEMs (Gel Bead-In EMulsion) were transferred to a new tube and placed at the C1000 Touch Thermal Cycler for the first phase of reverse transcription. All gene expression libraries were prepared and sequenced together to avoid batch effect. Deep sequencing was performed by the USC Molecular Genomics Core, using the Illumina NovaSeq6000 platform. At least 40,000 reads were obtained per cell.

Barcode specifically labeled the transcripts of each signal cell by using a Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics Inc, San Francisco, USA). Libraries were constructed and sequenced on an Illumina NovaSeq 6000 platform (S10OD032203) at a sequencing depth of ~800 million reads in Tufts University Core Facility (TUCF). The sequencing results were demultiplexed and cell barcodes were extracted. Subsequently, Single-Cell Analysis in Python (SCANPY) 26 (link) was utilized for data clustering analysis and visualization. SCSA 29 , an automatic tool and distinct marker genes from published papers was used for cluster annotation. The ligand-receptor interaction data were generated by Squidpy 58 (link) (based on CellPhoneDB 59 (link) and Omnipath 60 (link) database) via screening and paring the ligands and receptors, which expressed in more than 10% of cells in each cluster. The lncRNA-RNA reaction tool, intaRNA 52 (link),71 (link), was used to predict the direct bind site of lncR-APDC and Tff2. The newest intaRNA package was downloaded from GitHub to perform the analysis, which was able to analyze more than 2000 nt full length of lncRNAs and can be optimized on multiple settings.

By using Chromium Next GEM Single Cell 3′ Kit v3.1 (10x Genomics, 1000268) and Chromium Next GEM Chip G Single Cell Kit (10xGenomics, 1000120), we performed single cell 3′ gene expression profiling. The cell suspension 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. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Cell-barcoded 3′ gene expression libraries were sequenced on an Illumina NovaSeq6000 system by Shanghai Biochip Co., Ltd., Shanghai, China.

scRNA-seq was performed using a Chromium Next GEM Single Cell 3′ kit v.3.1 (10x Genomics) following the manufacturer’s protocol. The number of cells targeted for each condition was 3,000. Resulting libraries were sequenced on a NovaSeq 6000 or HiSeq 4000 system (Illumina).

For Slide-tags snRNA-seq experiments, 43.3 µl of counted nuclei was loaded into the 10x Genomics Chromium controller using the Chromium Next GEM Single Cell 3′ Kit v3.1 (10x Genomics, PN-1000268). The Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (Dual Index) with Feature Barcode Technology for Cell Surface Protein CG000317 was used according to the manufacturer’s recommendations with slight modifications. Spatial barcode libraries were prepared as cell-surface protein library preparations. The number of PCR cycles used for the index PCR step in the cell-surface protein library preparation (step 4.1f) for 5.5 × 5.5 mm TAGS arrays was 7; for 3 mm diameter TAGS arrays the number of cycles was 9.
For the mouse brain sample, ligated pucks (see sequence in the ‘Barcoded bead synthesis, array fabrication and sequencing’ section) were used for spatial barcoding. For this sample, a custom PCR protocol was used instead of step 4.1: 10 μl of cleaned supernatant from step 2.3, 50 µl NEBNext High-Fidelity 2× PCR Master Mix (NEB, M0541S), 2.5 µl STAG_P701_NEX (10 μM), 2.5 µl 10 μM P5-Truseq Hybrid oligo and 35 µl ultrapure DNase/RNase-free distilled water (Invitrogen, 10977015). In this sample, ten PCR cycles were performed according to the manufacturer’s recommendations.