Cell suspensions from 400 to 1200 cells/μl were loaded into the Chromium Single Cell Controller (10x Genomics) to target a recovery of 7000 to 10,000 cells per sample. Single-cell Gel bead-in-EMulsions (GEMs) were generated, cells were lysed, and the released RNAs were retrotranscribed (RT) and barcoded inside the GEMs according to Single Cell 3′ Reagent Kits v2 and v3 (10x Genomics) protocols. Following GEM break, barcoded cDNAs were pooled and cleaned from leftover RT reagents using DynaBeads MyOne Silane Beads (Invitrogen) and amplified (8 to 14 cDNA amplification cycles) following the Single Cell 3′ Reagent Kits v2 and v3 (10x Genomics) protocols. Enzymatic fragmentation, size selection, and cleanup with SPRIselect Reagent kit (Beckman Coulter) were followed by library construction (total sample index cycles from 12 to 16 cycles), consistent with standard Illumina sequencing constructs and performed in line with Single Cell 3′ Reagent Kits v2 and v3 (10x Genomics) protocols. cDNA and library concentration and quality check were assessed using Bioanalyzer High Sensitivity DNA Kit (Agilent) and 2100 Expert Software (Agilent). Libraries for 18 samples (E34VZSG, n = 3; E34VZLS, n = 3; P1VZSG, n = 3; P1VZLS, n = 3; P1OSVZSG, n = 3; P1OSVZLS, n = 3) were generated.
Single cell 3 reagent kit
Manufactured by 10x Genomics
Sourced in United States
The Single Cell 3' Reagent Kit is a laboratory equipment product from 10x Genomics. It is designed for single-cell RNA sequencing applications. The kit includes the necessary reagents to perform the library preparation steps for generating 3' gene expression data from individual cells.
Automatically generated - may contain errors
Lab products found in correlation
Chromium controller, by 10x Genomics (5 mentions)
Gemcode single cell instrument, by 10x Genomics (3 mentions)
Chromium system, by 10x Genomics (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Nextseq 500 platform, by Illumina (2 mentions)
Bioanalyzer high sensitivity dna kit, by Agilent Technologies (2 mentions)
Chromium single cell controller, by 10x Genomics (2 mentions)
I7 multiplex kit, by 10x Genomics (2 mentions)
Novaseq 6000 system, by Illumina (2 mentions)
Gemcode instrument, by 10x Genomics (1 mentions)
Cell ranger pipeline, by 10x Genomics (1 mentions)
Seqgeq software, by FlowJo (1 mentions)
Loupe browser, by 10x Genomics (1 mentions)
Novaseq platform, by Illumina (1 mentions)
Cell ranger 3, by 10x Genomics (1 mentions)
Nextseq 500 550, by Illumina (1 mentions)
Hiseq 2500 v4, by Illumina (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Facsaria 2 cytometer, by BD (1 mentions)
23 protocols using single cell 3 reagent kit
1
Single-cell RNA-seq Library Preparation
2
Isolation and scRNA-seq of Notch-lineage lung cells
3
Single-cell Transcriptome Profiling and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell capture and pre-amplification were conducted on a GemCode instrument (10× Genomics) according to the manufacturer’s ‘instructions (Chromium™ Single Cell 3’ Reagent Kit v2). The generated library was sequenced using the Illumina X10 platform and the generated sequencing reads were aligned and analyzed using the Cell Ranger Pipeline (10× Genomics). The raw count data of each single cell were deposited into the public database of the Genome Sequence Archive for Human (GSA-Human) under accession number HRA000928. Single-cell analysis was conducted using Seurat9 (link). The potential doublet of single-cell data was detected using DoubleFinder10 (link). A connectivity map was constructed according to a previous ligand-receptor dataset35 (link) using CellPhoneDB16 (link). GO analysis was conducted using http://geneontology.org . Gene set variation analysis (GSVA) was used to perform deconvolution analysis36 .
4
Single-cell transcriptome profiling of atherosclerosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anticoagulated peripheral blood collected from patients with atherosclerotic disease or matched healthy controls were used for the isolation of PBMCs by density gradient centrifugation using Ficoll as previously described.[21c ] Isolated PBMCs were loaded into single‐cell gel beads and barcoded with unique molecular identifier using the single cell 3’ reagent kit (10× Genomics) as instructed by the manufacturer's protocol. The complementary DNA (cDNA) was then generated, libraries were constructed, and sequenced on a NovaSeq platform (Illumina). The data was analyzed by Loupe Browser (10× Genomics) and SeqGeq Software (FlowJo). For the validation, single cell RNA sequencing datasets from PlaqView[10 ] were reanalyzed to detect the expression of Dpp4 in human aorta/plaque‐infiltrating cells from patients with atherosclerosis,[9a ] aortic aneurysm, and control subjects.[9b ]
5
Transcriptome Profiling of Malignant Ascites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Massively parallel droplet-based data from malignant ascites of AGC04 patient was generated to target 5000 cells using the Chromium System (10× Genomics, Pleasanton, CA, USA) with the Single Cell 3′ Reagent Kit (v2) following the manufacturer’s instructions. Libraries were sequenced on the HiSeq 2500 system, and reads were aligned to the GRCh38 human reference genome using Cell Ranger v2.1 software with default options.
Expression matrix processing, cell clustering, and dimensionality reduction were performed using Seurat v3.0. The gene Es for gene i in cell j was defined as Eij = log2(pseudoTPMij + 1), where pseudoTPMij = UMIij/sum[UMItotal,j] * 10,000. Cells were clustered by the graph-based shared nearest-neighbor (SNN) method, and cells in cluster 0 and 4 that exhibited differentially expressing macrophage markers (such as CD68) were defined as macrophages. Low-quality cells that did not meet any of the following criteria were excluded from the macrophage analysis: (1) 1000 ≤ nCounts ≤ 150,000, (2) 200 ≤ nFeatures ≤ 10,000, and (3) percent of mitochondrial genes ≤ 20.
Expression matrix processing, cell clustering, and dimensionality reduction were performed using Seurat v3.0. The gene Es for gene i in cell j was defined as Eij = log2(pseudoTPMij + 1), where pseudoTPMij = UMIij/sum[UMItotal,j] * 10,000. Cells were clustered by the graph-based shared nearest-neighbor (SNN) method, and cells in cluster 0 and 4 that exhibited differentially expressing macrophage markers (such as CD68) were defined as macrophages. Low-quality cells that did not meet any of the following criteria were excluded from the macrophage analysis: (1) 1000 ≤ nCounts ≤ 150,000, (2) 200 ≤ nFeatures ≤ 10,000, and (3) percent of mitochondrial genes ≤ 20.
6
Multimodal Single-Cell Analysis of Irradiated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells at stage prior to budding, when budding occurs and after budding were prepared for 10× scRNA‐seq and two samples named “Mix1” and “Mix2” were loaded onto an independent 10×Chrominum instrument (10× Genomics) per the user instructions. In brief, 8Gy‐irradiated HCT116 cells in different stages constituting group IR were collected for “Mix1”, followed by the assessment of cellular viability and adjustment of concentration to 1000 µL−1. Before the same process of preparation for “Mix2”, group of NC, IR and R cells were incubated with individual antibody tags (Biolegent TotalSeq) to be distinguishable from each other. An input of estimated 10000 cells was transfer to each channel. Single‐cell libraries were constructed using the Single Cell 3’ Reagent Kit (10× Genomics, v3.1). Libraries were then performed quality control using Bioanalyzer High Sensitivity DNA kit (Agilent) and sequenced on an Illumina NextSeq 500 platform (Genergy Inc.).
7
Single-Cell RNA-Seq of Tumor Cell Dissociation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 184-hTERT cells were pelleted and gently resuspended in 200 µl PBS followed by 800 µl 100% methanol and incubation at −20 °C for 30 min to fix, dehydrate and shrink cells. PDX tumour fragments were dissociated into single cells using collagenase/hyaluronidase at 37 °C for 2 h for TNBC tumours or with cold active Bacillus lichenformis (Creative Enzymes NATE0633) in PBS supplemented with 5 mM CaCl2 and 125 U ml−1 DNAse for HGSC tumours, as described previously45 (link) with additional mechanical dissociation using a gentleMACS dissociator (Miltenyi Biotec). Cells were then pelleted and resuspended in 0.04% BSA/PBS and immediately loaded onto a 10X Genomics Chromium single-cell controller targeting 3,000 cells for recovery. Libraries were prepared according to the 10X Genomics Single Cell 3′ Reagent kit standard protocol. Libraries were then sequenced on an Illumina Nextseq500/550 with 42-bp paired-end reads, or a HiSeq2500 v4 with 125-bp paired-end reads. 10X Genomics Cell Ranger 3.0.2 was used to perform demultiplexing, counting and alignment to GRCh38 and mm10.
8
Isolation and scRNA-seq of Notch-lineage lung cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For lineage-labelled cells from Red2-NotchN1ICD mice, YFP+CD45–CD31–EpCAM+ or RFP+CD45–CD31–EpCAM+ cells were sorted at day 28 post bleomycin injury (4 mice were pooled for each experiment). For non-lineage-labelled cells isolated from Red2-NotchN1ICD mice in parallel with experiment of lineage-labelled cells, we combined the cells of EpCAM+RFP–YFP– and EpCAM– population with a ratio of 1:1, respectively. The resulting cell suspension were submitted as separate samples to be barcoded for the droplet-encapsulation single-cell RNA-seq experiments using the Chromium Controller (10X Genomics). Single cell cDNA synthesis, amplification, and sequencing libraries preparation were performed using the Single Cell 3’ Reagent Kit as per the 10x Genomics protocol.
9
Single-Cell RNA-Seq of Murine Aortic Immune Cells
10
Single-cell RNA-seq of CD45-CD24- cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single CD45-CD24- cells were sorted by FACS ARIA III (BD) and collected in PBS containing 0.04% w/v BSA at a density of 400 cells/μl. Chromium™ Controller was used for partitioning single cells into nanoliter-scale Gel Bead-In-EMulsions (GEMs) and Single Cell 3’ reagent kit v2 for reverse transcription, cDNA amplification and library construction (10xGenomics, Cat. #120236). The detailed protocol was provided by 10xGenomics. SimpliAmp Thermal Cycler was used for amplification and incubation steps (Applied Biosystems). Libraries were quantified by QubitTM 3.0 Fluometer (ThermoFisher) and quality checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent). Sequencing was performed in paired-end mode (2 × 75 cycles) on an Illumina NextSeq 500 sequencer to attain approximately 75,000 ± 25,000 reads per single cell. The scRNA-seq raw and processed data have been deposited in the NCBI GEO database under accession code GSE172526 and GSE106489 for D56 mLN-SPF11 (link).
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!