Organoids were harvested, washed in PBS, pelleted, and frozen on dry ice for snRNA-seq. Nuclei were isolated following the protocol described previously142 (link). Briefly, frozen samples were resuspended in TST buffer (146 mM NaCl, 10 mM Tris, 1 mM CaCl2, 21 mM MgCl2, 0.03% Tween20, and BSA) and pelleted by centrifugation. The nuclei pellets were resuspended in ST buffer (146 mM NaCl, 10 mM Tris, 1 mM CaCl2, 21 mM MgCl2), passed through a 35 µm filter, and counted using a hemocytometer. Generation of gel Beads-in-emulsion, cDNA amplification, and library preparation were performed with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics) following the manufacturer’s instructions. 8000-10,000 nuclei were loaded per channel of a 10x Genomics chip.
Chromium next gem single cell 3 reagent kits v3
Manufactured by 10x Genomics
Sourced in United States
The Chromium Next GEM Single Cell 3' Reagent Kits v3.1 are a set of laboratory reagents designed for single-cell RNA sequencing. The kits enable the capture, barcoding, and library preparation of individual cells for subsequent analysis using 10x Genomics' Chromium platform.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (12 mentions)
Chromium next gem chip g, by 10x Genomics (7 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (6 mentions)
4200 tapestation system, by Agilent Technologies (6 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (6 mentions)
Chromium controller, by 10x Genomics (4 mentions)
Nextseq 500 machine, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Novaseq 6000 system, by Illumina (3 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (3 mentions)
Nextseq 500 system, by Illumina (3 mentions)
Qubit 2.0 fluorometer with kit high sensitivity assays, by Agilent Technologies (3 mentions)
Nextseq 550 system high output kit, by Illumina (3 mentions)
Single cell rna seq kit, by 10x Genomics (2 mentions)
Novaseq instrument, by Illumina (2 mentions)
Novaseq, by Illumina (2 mentions)
D1000 dna screentape, by Agilent Technologies (2 mentions)
Tapestation 4200, by Agilent Technologies (2 mentions)
Nextseq 2000, by Illumina (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
48 protocols using chromium next gem single cell 3 reagent kits v3
1
Nuclei Isolation for snRNA-seq
2
Single-Cell RNA Sequencing of Tumor Cells
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Tumors were finely minced at 4°C and transferred into tumor digestion medium containing collagenase/hyaluronidase and DNase I in RPMI 1640 with glutamine, then incubated on a shaker for 45 min at 37C and 300rpm. Freed cells were collected by passing through the dissociated tumor and media into a 70um cell strainer and quenching with FACS buffer (2% fetal bovine serum in sterile PBS) at 4°C. Cells were spun down at 300g for 5 min at 4°C, the pellet resuspended in ice-cold ammonium chloride solution for 5 min and quenched with FACS buffer. Cells were spun down again and resuspended in FACS buffer. Viable cells were quantified by trypan blue method and samples were then subject to dead cell removal (EasySep Dead Cell Removal Kit, STEMCELL). Prior to library preparation, viability and cell number were re-assessed with a Countess II FL using AOPI (PN- CS2-0106-5mL, Nexcelom Bioscience). Single-cell RNA-seq libraries targeting 8,000 cells per sample were generated using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (PN-1000121, 10x Genomics) according to the manufacturer’s instructions. Final libraries were sequenced to at least 25,000 reads per cell with the Illumina NextSeq 2000 and aligned with Cell Ranger (version 6.0.0, 10x Genomics) to the mm10 reference genome (refdata-gex-mm10-2020-A, 10x Genomics).
3
Single-cell RNA-seq library preparation
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The cell concentration of fresh cell suspension for each sample was adjusted to 700–1200 cells/µL. Then the cell suspension was subjected to Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics, Pleasanton, CA) for library preparation according to the standard protocols. The single cell libraries were sequenced on Illumina NovaSeq 6000 Systems using paired‐end sequencing (150 bp). The Cell Ranger software pipeline (version 3.1.0) provided by 10x Genomics was used to demultiplex cellular barcodes, map reads to the genome and transcriptome using the STAR aligner, and down‐sample reads as required to generate normalized aggregate data across samples, producing a matrix of gene counts versus cells.
4
Single-cell RNA-seq of Dissociated Lung Cells
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Dissociated lung cells were stained and sorted for CD45−/GFP+ and CD45−/GFP− populations. Single-cell gene expression libraries were generated from the sorted cell suspensions using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v.3.1 (10X Genomics, Pleasanton, CA), loading an estimated 10,000 cells per sample and following the manufacturer’s instructions. Libraries were quantified with the KAPA Library Quantification Kit (Roche) and profiled using the Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA). Each library was sequenced in one lane of a HiSeq 4000 (Illumina, Foster City, CA) to generate 300 million paired-end reads at a configuration of 28 base pairs (read 1) and 98 base pairs (read 2). Sequencing reads were processed through 10X Genomics Cell Ranger (v.3.1.0) and aligned to GRCm38 with added sequences to measure EGFP reporter gene. We used the R package Seurat for downstream analysis. Cells with fewer than 200 genes expressed or more than 8,000 genes expressed were filtered out. Cells with 15% or more mitochondrial content were also removed. The Seurat function IntegrateData was used for data integration of the two populations. Clustering analysis was performed using FindClusters with a resolution set to 0.5.
5
Single-cell RNA-seq using 10x Genomics
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We used the 10x Genomics single-cell RNA-seq kit v3 to sequence barcoded cells. We resuspended the cells (aiming for up to 10,000 cells for recovery/ sample) in PBS and followed the protocol for the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 as per manufacturer directions (10x Genomics). Briefly, we generated gel beads-in-emulsion (GEMs) using the 10x Chromium system, and subsequently extracted and amplified barcoded cDNA as per post-GEM RT-cleanup instructions. We then used a fraction of this amplified cDNA (25%) and proceeded with fragmentation, end-repair, poly A-tailing, adapter ligation, and 10x sample indexing per the manufacturer’s protocol. We quantified libraries using the High Sensitivity dsDNA kit (Thermo Fisher #Q32854) on Qubit 2.0 Fluorometer (Thermo Fisher #Q32866) and performed Bioanalyzer 2100 (Agilent #G2939BA) analysis prior to sequencing on a NextSeq 500 machine (Illumina) using 28 cycles for read 1, 55 cycles for read 2, and 8 cycles for i7 index.
6
Single-Cell RNA-seq of Retinal Cells
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Single-cell RNA-sequencing and snRNA-seq were performed on dissociated retinal cells or nuclei using the Chromium Next GEM Single-Cell 3ʹ Reagent Kits v3.1 (10× Genomics). Briefly, retinal cells or nuclei (∼16 000 cells per sample) were loaded into the 10× Chromium controller and downstream scRNA-seq or snRNA-seq libraries were generated and indexed by following the manufacturer’s instructions. Libraries were pooled and sequenced on Illumina NovaSeq 6000 targeting 50 000 reads per cell.
7
Single-Cell Sequencing of Tumor Samples
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Tumors were collected into heparin tubes (Becton, Dickinson and Co.) and processed using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics; Pleasanton, CA, USA) according to the manufacturer’s instructions as described in our previous study.61 (link)
8
Chromium Next GEM Single Cell 3' Sequencing
9
Single-Cell RNA Sequencing with Chromium
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Single-cell libraries were prepared using Chromium NextGEM Single Cell 3ʹ Reagent Kits v3.1 (10X Genomics, Pleasanton, CA). The cells and kit reagents were mixed with gel beads containing the GEMs. The barcoded cDNAs in each GEM were pooled for PCR amplification, and adapter and sample indices were added after fragmentation targeting the 3ʹ end of the RNA. The generated libraries were paired-end sequenced on a HiSeq 2500 (Illumina, San Diego, CA) instrument, using the following read length: 28 bp Read1, 8 bp I7 index, and 91 bp Read2.
10
Single-cell RNA-seq Library Preparation
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scRNA-seq libraries were prepared according to the manufacturer’s instructions of Beijing SeekGene BioSciences Co., Ltd (Beijing, China). Libraries were prepared using Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10× Genomics). The appropriate number of cells were mixed with reverse transcription reagents and then loaded to the sample well in a Chromium Next GEM Chip G. Gel Beads and Partitioning Oil were then dispensed into corresponding wells, separately in a chip. We then performed emulsion droplet generation reverse transcription at 53 °C for 45 min and inactivated at 85 °C for 5 min. The cDNA was then purified from the broken droplet and amplified in a PCR reaction. The amplified cDNA products were then cleaned, fragmented, end-repaired, A-tailed, and ligated to the sequencing adapter. Finally, the indexed PCR was performed to amplify the DNA representing 3ʹ polyA part of expressing genes which also contained Cell Bar code and Unique Molecular Index. The indexed sequencing libraries were cleaned with SPRI beads, quantified by quantitative PCR (KAPA Biosystems KK4824), and sequenced on an Illumina NovaSeq 6000 with PE150 read length.
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