Libraries were generated using the Chromium Single Cell 5′ Library and Gel Bead Kit v1.1 (10x Genomics, Pleasanton, CA) following the manufacturer’s instructions. In short, pooled cells were loaded aiming the capture of 10,000 cells per pool. Libraries were generated according to standard protocol (Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 rev E). The amplified mRNA and antibody-derived tags were divided by size and sequenced separately.
Chromium single cell 5 library and gel bead kit v1
Manufactured by 10x Genomics
The Chromium Single Cell 5' Library and Gel Bead Kit v1.1 is a laboratory equipment product that enables the generation of single-cell 5' libraries for use in genomic analysis. The kit provides the necessary components to capture, barcode, and amplify RNA from individual cells.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000 system, by Illumina (1 mentions)
Chromium controller, by 10x Genomics (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Chromium i7 multiplex kit, by 10x Genomics (1 mentions)
Novaseq, by Illumina (1 mentions)
Genomics chromium controller, by 10x Genomics (1 mentions)
Chromium single cell a chip kit, by 10x Genomics (1 mentions)
Highseq 4000, by Illumina (1 mentions)
Tapestation 4200, by Agilent Technologies (1 mentions)
Countess 2 automated cell counter, by Thermo Fisher Scientific (1 mentions)
4 protocols using chromium single cell 5 library and gel bead kit v1
1
Single-cell transcriptome and antibody analysis
2
Single-Cell RNA Sequencing Workflow
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The single cells were loaded (aiming at 5,000 cells/sample for each chip position) onto and barcoded with a 10× Chromium Controller (10× Genomics) followed by library construction for scRNA-seq using the Chromium Single Cell 5′ Library and Gel Bead kit (V1.1) from 10× Genomics according to the manufacturer’s instructions. The 5′ gene expression libraries were sequenced on a NovaSeq 6000 system (Illumina) until sufficient saturation was reached (77.03% on average). Fastq alignment was performed in Cellranger 6.0.2 with GRCh38 as transcriptome reference.
3
Single-Cell RNA-Seq of Mesp1 KO Cells
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Libraries for scRNA-seq were prepared according to manufacturer's instructions using the 10x Genomics Chromium controller, Chromium Single Cell 5′ Library and Gel Bead Kit v1 (10x Genomics, 1000006) and Chromium Single Cell A Chip Kit (10x Genomics, 1000151). A targeted maximum recovery of 10,000 cells per sample were loaded onto the 10x Genomics Chromium instrument, and each sample was indexed with a unique sample identifier (10x Genomics Chromium i7 Multiplex Kit, 120262). Final libraries were pooled and sequenced shallowly according to 10x protocol parameters on a NextSeq500 (Illumina), and then re-pooled for deeper sequencing on HighSeq4000 (Illumina) and/or NovaSeq using an S4 lane (Illumina). Littermate, stage-matched comparisons of control and Mesp1 KO libraries were always sequenced together in the same library pool. All scRNA-seq libraries were sequenced to a mean read depth of at least 50,000 total aligned reads per cell.
4
Isolation and Sequencing of Single-Cell Nuclei
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Nuclei were isolated from the biopsies using the Singulator instrument (S2 Genomics). The instrument was primed with cold nuclei isolation and storage buffers (S2 Genomics) and the biopsy was loaded into the cartridge and covered with the 19.7 mm grinding cap. The “Nuclei_All_Tissues” protocol was used to isolate nuclei, after which the nuclei were centrifuged for 5 minutes and resuspended in buffer. The nuclei were counted using a Countess II Automated Cell Counter (Invitrogen), centrifuged again for 5 minutes, and resuspended at the proper concentration for use in the Chromium Single Cell 5’ Library and Gel Bead Kit v1 (10× Genomics). Samples were processed using the Chromium Single Cell A Chip Kit and Chromium Controller (10× Genomics). Quality control was performed on the Tapestation 4200 (Agilent Technologies). Libraries were sequenced in one lane of a NovaSeqS4 by the U.C. Davis Genomics Core.
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