Droplet-based scRNA-seq datasets were produced using a 10x Genomics Chromium system. B cells isolated from PP GCs were sorted into 80% methanol/PBS and 15,000 cells were loaded onto the Chromium™ Controller instrument following the manufacturer’s recommendations. Cells were partitioned into Gel Beads in Emulsion in the Chromium™ Controller instrument where cell lysis and barcoded reverse transcription of RNA occurred. Libraries were prepared using 10x Genomics Library Kits and sequenced on an Illumina NextSeq500 according the manufacturer’s recommendations. Raw sequencing files were aligned to the mouse V(D)J sequence using Cell Ranger: V(D)J Pipelines (10x Genomics) and the V usage and clonotype profiles were generated and visualized by Loupe V(D)J Browser. Note that due to low viability of GC B cells after isolation in vitro, which is critical for cell recovery according to 10x genomics manufacture’s protocol, we were able to recover only 600~1200 cells for each sample. The low cell recovery rate and low yield of scRNA-seq for GC B cells may introduce some variability in profiling the GC BCR repertoire.
Genomics chromium system
Manufactured by 10x Genomics
Sourced in United States
The 10x Genomics Chromium System is a lab equipment used for single-cell analysis. It enables the isolation, barcoding, and sequencing of individual cells from a sample. The system provides a platform for researchers to study gene expression, cellular diversity, and other genomic features at the single-cell level.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 4000, by Illumina (3 mentions)
Nextseq 500, by Illumina (2 mentions)
Noveseq 6000, by Illumina (2 mentions)
Genomics reagents, by 10x Genomics (2 mentions)
Hiseq 3000 system, by Illumina (2 mentions)
50 cycle sbs kits, by Illumina (1 mentions)
Hiseq 3000 4000 pe cluster kit, by Illumina (1 mentions)
Spriselect beads, by Beckman Coulter (1 mentions)
Chromium single cell 3 library gel bead kit v2, by 10x Genomics (1 mentions)
Hiseq x sequencer, by Illumina (1 mentions)
Real time analysis 2, by Illumina (1 mentions)
Loupe 2, by 10x Genomics (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Cell ranger single cell software suite, by 10x Genomics (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Chromium instrument, by 10x Genomics (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Novaseq sequencing system, by Illumina (1 mentions)
Cell ranger v6, by 10x Genomics (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
15 protocols using genomics chromium system
1
Single-cell RNA-seq of Germinal Center B Cells
2
Single-cell transcriptome profiling using 10x Genomics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspensions were processed through the 10 × Genomics Chromium System (10 × Genomics, Pleasanton, CA, USA), followed by the construction of 3’ gene expression v3.1 libraries and sequencing on an Illumina Noveseq6000 sequencer. Briefly, cells (> 90% viability), reagents, gel beads, and partitioning oil are loaded onto 10 × Chromium Chip B. Ideally, each individual cell is wrapped with an oil drop that contains gel beads and reagents. This creates an independent reaction space for each cell called gel bead in emulsion (GEM). The primers provided by the gel bead contain a sequencing primer, barcode, UMI, and poly (dT) sequence. The cell in the GEM is lysed, and incubation of the GEM produces barcoded, full-length cDNA from poly-adenylated mRNA. Next, all cDNAs are pooled together, and the steps of general library construction are performed including amplification, fragmentation, end repair, adapter ligation, and sample index polymerase chain reaction (PCR) (98 °C for 45 s; [98 °C for 20 s, 67 °C for 30 s, and 72 °C for 1 min] × 14 cycles; 72 °C for 1 min). The libraries were then sequenced using Illumina Noveseq6000 by CapitalBio Technology (Beijing, China). Sequencing data are available at Genome Sequence Archive (accession no. HRA005871).
3
Scalable scRNA-seq Analysis using 10x Genomics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 10× Genomics Chromium system and Single Cell 3’ Reagent v2 Kits (10× Genomics, Pleasanton, CA) were used to generate scRNA-seq libraries according to the manufacturer’s instructions. The reverse transcribed cDNA was first amplified by 14 PCR cycles and then used to generate the sequencing library, which was finally amplified by 14 PCR cycles. The sequencing library was purified as described by the manufacturer and then subjected to another cleanup step using one volume of SPRISelect Beads (Beckman Coulter, Brea, CA, USA) in order to remove leftover primers and dimers. The average length of the purified library was 494 bp. A HiSeq 4000 and two 50-cycle SBS kits (Illumina, San Diego, CA, USA) were used for sequencing and clusters were generated on a cBot using the HiSeq 3000/4000 PE Cluster Kit. The paired-end sequencing run comprised a 26-bp read 1 (16 bp cell barcode and 10 bp UMI) and 98-bp read 2 (transcript sequence read) as well as a 8-bp index read. On average, approximately 50,000 reads were sequenced per cell.
4
Single-cell RNA-sequencing with 10x Genomics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspensions were processed through the 10× Genomics Chromium System (10× Genomics, Pleasanton, CA), followed by the construction of 3′ gene expression v3.1 libraries and sequencing on an Illumina Noveseq6000 sequencer. Briefly, cells (>90% viability), reagents, gel beads, and partitioning oil are loaded onto 10× Chromium Chip G. Ideally, each individual cell is wrapped with an oil drop that contains gel beads and reagents. This creates an independent reaction space for each cell called gel bead in emulsion. The primers provided by the gel bead contain a sequencing primer, barcode, unique molecular identifier (UMI), and poly (dT) sequence. The cell in the gel bead in emulsion is lysed, and incubation of the gel bead in emulsion produces barcoded, full-length cDNA from polyadenylated mRNA. Next, all cDNAs are pooled together, and the steps of general library construction are performed including amplification, fragmentation, end repair, adapter ligation, and sample index polymerase chain reaction (98 °C for 45 s [98 °C for 20 s, 67 °C for 30 s, and 72 °C for 1 minute]×14 cycles; 72 °C for 1 minute). The libraries were then sequenced using Illumina Noveseq6000.
5
Single-cell RNA-seq Library Preparation
6
Single-cell RNA-seq of tumor-immune interactions
7
Haplotype-Phasing through Synthetic Long-Read Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Haplotype-phasing of the patient and his father were performed through synthetic long-read whole genome sequencing (Macrogen, Seoul, South Korea). Genomic DNA samples were extracted from leukocytes. Sequencing libraries were prepared using the 10x Genomics Chromium System (10x Genomics, Pleasanton, CA, USA) and loaded on the HiSeqX sequencer (Illumina). Sequence data were obtained as 150-bp paired-end reads. Base calling and quality scoring were performed using Real Time Analysis 2 (Illumina). The base call files were converted to FASTQ files using the Long Ranger 2.1.3 (10x Genomics). Then, Long Ranger 2.1.5 (10x Genomics) was used to align sequence reads and to call single nucleotide variants (SNVs), indels, and structural variants. The output data were visualized on the Loupe 2.1.2 (10x Genomics). We genotyped SNVs of the patient and his father and constructed haplotype-phase blocks. In this analysis, we focused primarily on genomic regions flanking the breakpoints.
8
Single-cell Transcriptome Profiling with 10x
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The single-cell library preparation relied on a commercially available droplet method, the 10x Genomics Chromium System (10x Genomics Inc San Francisco, CA). The number of cells loaded on the system was calculated based on the desired number of captured cells following manufacturer’s instructions. scRNA-seq libraries were generated following capture. Chromium GFP+ day 3 samples were sequenced on the Illumina HiSeq 2500 and the remainder on the Illumina NextSeq 500 high output.
9
Single-cell RNA-seq of Human and Chimpanzee μPASEs
10
Single-cell RNA-seq of tumor-immune interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Biological replicates from n=3 pooled mice were processed for each
experimental condition from YUMMER1.7 and
YUMMER1.7-B2m−/− tumors.
Biological replicates were then pooled together at the single cell suspension
stage with equivalent number of cells from each replicate. The following
populations were sorted purified: P1:
GFP−CD45+CD3+ (T cells), P2:
GFP−CD45+CD3− (non-T immune
cells), P3:
GFP+/−CD45−CD3− (tumor
and stromal cells). P1, P2, and P3 for each sample were then mixed back together
at a 2:1:1 ratio, respectively. 5000 cells from each of the mixed sorted samples
for each condition were loaded onto the 10x Genomics Chromium System. Library
preparation was performed using 10x Genomics reagents according to the
manufacturer’s instructions and was performed by the Yale Center for
Genome Analysis (YCGA) and passed QC. Libraries were sequenced using an Illumina
HiSeq 4000 (one library/lane) at the YCGA.
experimental condition from YUMMER1.7 and
YUMMER1.7-B2m−/− tumors.
Biological replicates were then pooled together at the single cell suspension
stage with equivalent number of cells from each replicate. The following
populations were sorted purified: P1:
GFP−CD45+CD3+ (T cells), P2:
GFP−CD45+CD3− (non-T immune
cells), P3:
GFP+/−CD45−CD3− (tumor
and stromal cells). P1, P2, and P3 for each sample were then mixed back together
at a 2:1:1 ratio, respectively. 5000 cells from each of the mixed sorted samples
for each condition were loaded onto the 10x Genomics Chromium System. Library
preparation was performed using 10x Genomics reagents according to the
manufacturer’s instructions and was performed by the Yale Center for
Genome Analysis (YCGA) and passed QC. Libraries were sequenced using an Illumina
HiSeq 4000 (one library/lane) at the YCGA.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!