Cells originating from human thyroid differentiation protocol, at day 45 and day 58, were isolated for scRNAseq profiling, following the procedures previously described37 . Single-cell suspension preparation and FACS cell sorting were performed as previously mentioned (“NKX2-1GFP population expansion assessment and proliferation assay - flow cytometryn ” and RNA-seq and analysis of bulk samples sections). Different proportions of viable NKX2-1/GFP+ (60%) and NKX2-1/GFP− (40%) cells were sorted to guarantee the representation of the distinct cell types present in the organoid culture. Sorted cells were collected in PBS at a density of 800 cells/μl and diluted accordingly to kit’s instruction (10x Genomics Chromium Single Cell 3′ v3). Around 6000 cells were loaded onto a channel of the Chromium Single Cell 3′ microfluidic chip and barcoded with a 10X Chromium controller followed by RNA reverse transcription and amplification according to manufacturer’s recommendations (10X Genomics). Library preparation was performed based on 10X Genomics guidelines. Libraries were sequenced using Illumina NovaSeq 6000 system.
Chromium single cell 3 v3
Manufactured by 10x Genomics
Sourced in United States
The Chromium Single Cell 3' v3 is a lab equipment product from 10x Genomics. It is a system designed for single-cell RNA sequencing, allowing researchers to analyze gene expression profiles at the individual cell level.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (2 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
Chromium controller, by 10x Genomics (1 mentions)
Papain dissociation protocol, by Worthington (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Sytox blue viability dye, by Thermo Fisher Scientific (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Mouse tumor dissociation kit, by Miltenyi Biotec (1 mentions)
Bcl2fastq2, by Illumina (1 mentions)
Dulbecco s modified, by Thermo Fisher Scientific (1 mentions)
Novaseq, by Illumina (1 mentions)
Dnase 1, by Roche (1 mentions)
Collagenase a, by Roche (1 mentions)
Totalseq b0301 b0306, by BioLegend (1 mentions)
Hyaluronidase, by Worthington (1 mentions)
Novaseq 6000 s4, by Illumina (1 mentions)
Cell ranger, by 10x Genomics (1 mentions)
8 protocols using chromium single cell 3 v3
1
Transcriptomic Profiling of Thyroid Organoids
2
Single-cell RNA-seq library construction
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The single-cell capturing and downstream library constructions were performed using the Chromium Single Cell 3' v3 (10x Genomics) library preparation kit according to the manufacturer’s protocol. Cellular suspensions were co-partitioned with barcoded gel beads to generate single-cell gel bead-in-emulsion (GEM) and polyadenylated transcripts were reverse-transcribed. Incubation of the GEMs produces barcoded, full-length cDNA from polyadenylated mRNA, and amplified via PCR to generate sufficient mass for library construction. Then, the libraries were sequenced on NovaSeq6000 (Illumina).
3
Papain Dissociation for Single-cell RNA-seq
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We collected three organoids per samples and dissociated using the papain dissociation protocol (Worthington Biochemicals) according to the manufacturer’s protocol. Briefly, organoids were incubated for 30 min at 37 °C in Earle’s Balanced Salt Solution (EBSS)/Albumin-ovomucoid inhibitor/papain with gentle shaking and mechanical dissociation. The pellet was resuspended and incubated with (EBSS)/Albumin-ovomucoid inhibitor/DNAse for 2 min followed by incubation with ovomucoid for 2 min. The cells were washed and resuspended with DMEM/F12. After determination of the cell density and viability, cells were submitted to single-cell RNA sequencing (10X Genomics, Chromium Single cell 3’ v3) to recover ~5000 sequenced cells per sample with more than 50,000 reads per cell. We have uploaded the RNAseq data used in this paper in NCBI’s Gene Expression Omnibus under accession code GSE174569.
4
Circadian Skin Cell Isolation Protocol
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Immediately after sacrificing a mouse with CO2, hair on dorsal skin was removed with an electric razor and Nair Hair Removal cream. After the dorsal skin was isolated from the body, fat and remaining blood vessels were scrapped away with a scalpel blade. A circular piece of skin was obtained with a 12mm biopsy punch, and minced into tiny pieces. The minced skin was then digested with 2mL of a solution consisting of 0.27% Collagenase IV (Sigma, C5138), 10mM HEPES (Fisher Scientific, BP310-100), 1mM Sodium Pyruvate (Fisher Scientific, BP356-100), and 5U/mL DNase I (Thermo Scientific, EN0521) at 37 °C for 1.5 hours. The suspension was then filtered with 70 μm and 40 μm cell strainers to obtain single cells. SYTOX blue viability dye (1:1000; Invitrogen, S34857) was added to the cell suspension and live cells were sorted out using FACS at the UCI Stem Cell Research Center.
Samples were collected every four hours for three days to generate in total of 18 samples, providing three biological replicates per circadian time point. The Chromium Single Cell 3’ v3 (10x Genomics) libraries were prepared and sequenced by the University of California Irvine Genomic High Throughput Facility with Illumina NovaSeq6000.
Samples were collected every four hours for three days to generate in total of 18 samples, providing three biological replicates per circadian time point. The Chromium Single Cell 3’ v3 (10x Genomics) libraries were prepared and sequenced by the University of California Irvine Genomic High Throughput Facility with Illumina NovaSeq6000.
5
Elucidating CD276-Mediated Immune Escape in ESCC
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scRNA-seq was performed on ESCC tissues obtained from epithelial conditional knockout CD276 (K14CreER, Cd276flox/flox) mice and control mice (Cd276wt/wt) to elucidate the main mechanism by which CD276 regulates immune escape in ESCC. To prepare the single-cell suspension, esophageal tissues were microdissected and disaggregated into single cells using enzymatic digestion with mouse Tumor Dissociation Kit (130-096-730, Macs Miltenyi Biotec, China) at 37 °C for 45 min using gentleMACS Dissociator (130-093-235, Macs Miltenyi Biotec, China). For scRNA-seq library preparation, we used the Chromium Single Cell 3′ v3 (10× Genomics, Pleasanton, California, USA) following the manufacturer’s instructions.
Seurat R package was employed to analyze and visualize the scRNA-seq data.37 (link) Briefly, we identified high-variable genes, capturing heterogeneity and key regulatory factors. Next, principal component analysis reduced dimensionality, enabling visualization of cellular heterogeneity, and identifying cell clusters based on gene expression patterns.
Seurat R package was employed to analyze and visualize the scRNA-seq data.37 (link) Briefly, we identified high-variable genes, capturing heterogeneity and key regulatory factors. Next, principal component analysis reduced dimensionality, enabling visualization of cellular heterogeneity, and identifying cell clusters based on gene expression patterns.
6
Dissociation and Labeling of Murine Myc;Ptenfl Tumors
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Single-cell suspensions of 11 Myc;Ptenfl tumors from 6 mice were obtained by enzymatic digestion. Tissue was manually minced using scissors, followed by a 30–60 min enzymatic digestion with 2.0 mg/ml collagenase A (Roche), 1.0 mg/ml Hyaluronidase (Worthington), and 50 U/ml DNase I (Roche) in serum-free Dulbecco’s modified eagles medium (DMEM) (Invitrogen) and Rock inhibitor at 37 °C using continuous stirring conditions. Single-cell suspensions from tumor digests were prepared by passing tissue through 40-mm nylon strainers (BD Biosciences). Single-cell suspensions from individual tumors were then labeled with hashtag oligonucleotides following the manufacturer’s protocol (TotalSeq B0301–B0306, Biolegend). Each individual tumor sample was counted and then pooled at an equal cell ratio before being split into two replicates for library preparation with the Chromium Single Cell 3’ V3 (10× Genomics) following the manufacturer’s protocol with a targeted recovery of 20,000 cells per library. Libraries were sequenced on an Illumina NovaSeq. BCL files were converted to fastq format with bcl2fastq2 (Illumina) and then aligned to mouse genome build mm10-2020-A (10× Genomics) using Cellranger (10× Genomics, version 6.0.2).
7
Bulk scRNA-seq using 10x Chromium
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scRNA-seq libraries were generated using Chromium Single Cell 3′ v.3 (10x Genomics). Libraries were sequenced using NovaSeq 6000 S4 (Illumina) 2 × 100 bp paired-end reading strategy. Cells were called using Cell Ranger (v.6.1.2, 10x Genomics) with the mouse reference genome (mm10) to generate raw gene expression matrices for each sample.
8
Lymph Node Single-Cell Transcriptomics
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Pooled LNs were isolated from female WT and KO littermates aged D4-6, D12-15, and D30-45. Each replicate was processed, encapsulated, and sequenced independently. Samples were gently digested and cytofluorometrically sorted according to CD45.2 expression and encapsulated using the Chromium Single cell 3′ v3 (10× Genomics).
Data were processed using the standard cellranger pipeline (10× Genomics). Downstream analysis was performed using the Seurat v4 pipeline (56) . Velocity analysis was conducted using the scVelo package (57) . Cell-interaction networks were constructed using the cellchat package (29) . Further information related to data analysis can be found in SI Appendix, Materials and Methods.
Population-Level RNA-seq Library Preparation and Sequencing.
Flow cytometrically purified cell populations ranging from 500 to 1,000 cells were double-sorted for each replicate. Library preparation was conducted using the Smart-seq2 RNA-seq protocol. Sequencing was conducted at the Broad Genomics Platform following the standard Immgen (https://www.immgen.org). Further information on data processing and quality control can be found in SI Appendix, Materials and Methods.
Data were processed using the standard cellranger pipeline (10× Genomics). Downstream analysis was performed using the Seurat v4 pipeline (56) . Velocity analysis was conducted using the scVelo package (57) . Cell-interaction networks were constructed using the cellchat package (29) . Further information related to data analysis can be found in SI Appendix, Materials and Methods.
Population-Level RNA-seq Library Preparation and Sequencing.
Flow cytometrically purified cell populations ranging from 500 to 1,000 cells were double-sorted for each replicate. Library preparation was conducted using the Smart-seq2 RNA-seq protocol. Sequencing was conducted at the Broad Genomics Platform following the standard Immgen (https://www.immgen.org). Further information on data processing and quality control can be found in SI Appendix, Materials and Methods.
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