DRG collected from 3 mice from each genotype for two biological replicates were dissociated into single cell suspension as describe (Avraham et al., 2020 (link), 2021b (link)). Cells were then washed in HBSS + Hepes + 0.1%BSA solution, passed through a 70-micron cell strainer. Hoechst dye was added to distinguish live cells from debris and cells were FACS sorted using MoFlo HTS with Cyclone (Beckman Coulter, Indianapolis, IN). Sorted cells were washed in HBSS + Hepes + 0.1%BSA solution and manually counted using hemocytometer. Solution was adjusted to a concentration of 500 cell/microliter and loaded on the 10X Chromium system. Single-cell RNA-Seq libraries were prepared using GemCode Single-Cell 3’ Gel Bead and Library Kit (10x Genomics). A digital expression matrix was obtained using 10X’s CellRanger pipeline (Build version 2.1.0) (Washington University Genome Technology Access Center). Quantification and statistical analysis were done with Partek Flow package (Build version 9.0.20.0417).
Gemcode single cell 3 gel bead and library kit
Manufactured by 10x Genomics
The GemCode Single Cell 3' Gel Bead and Library Kit is a laboratory equipment product from 10x Genomics. It is designed to facilitate the preparation of single-cell RNA sequencing libraries.
Automatically generated - may contain errors
Lab products found in correlation
Gemcode single cell instrument, by 10x Genomics (3 mentions)
Nextseq 500, by Illumina (2 mentions)
Hiseq 2500, by Illumina (1 mentions)
2200 tapestation system, by Agilent Technologies (1 mentions)
Chromium single cell 3 library v3 kit, by 10x Genomics (1 mentions)
Chromium next gem single cell 3 gel beads v3.1 kit, by 10x Genomics (1 mentions)
Chromium system, by 10x Genomics (1 mentions)
6 protocols using gemcode single cell 3 gel bead and library kit
1
Single-cell RNA-Seq of Dissociated DRG Cells
2
Single-cell RNA-seq of B-cell Lineage
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Single-cell RNA-seq was performed according to the manufacturer's protocol as described previously (Zheng et al. 2017 (link)). Briefly, iLS cells at day 0 and day 8 of B-cell induction or LMPP, CLP, and pro-B cells in BM were sorted, and ∼3000 cells were loaded onto a GemCode single-cell instrument (10x Genomics) to generate single-cell gel beads in emulsion (GEMs). Single-cell RNA-seq libraries were prepared using GemCode single-cell 3′ gel bead and library kit (10x Genomics). Sequencing libraries were loaded onto an Illumina HiSeq 2500. Cell Ranger single-cell software was used to perform sample demultiplexing, barcode processing, and single-cell 3′ gene counting and to combine libraries. For secondary analysis, including t-SNE projection and clustering, Cell Ranger R kit was used.
3
Single-Cell RNA Sequencing of Plasmacytoid Dendritic Cells
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Briefly after pDCs were isolated, cells and reagents were prepared and loaded into the chip and ran into the Chromium Controller for Gel Bead-In Emulsion (GEM) generation and barcoding. The input number of cells was estimated at 15-20,000 cells per sample. The Chromium Next GEM Single Cell 3’ Gel beads v3.1 kit (10X Genomics, Pleasanton, CA, USA) was used to create GEMs following manufacturer’s instruction. All GEMs generated were used for cDNA synthesis and library preparation using the Chromium Single Cell 3’ Library Kit v3.1 (10X Genomics) following the manufacturer’s instruction. scRNA-seq libraries were then prepared using GemCode Single Cell 3′ Gel bead and library kit (10X Genomics) following the manufacturer’s instruction. cDNA concentration of each sample was measured using a Tapestation 2200 system (Agilent). Single-cell barcoded cDNA libraries were sequenced on an Illumina Illumina Novaseq6000 system (100-cycle cartridge) with a sequencing depth of at least 50,000 reads per cell.
4
Single-Cell RNA-Seq of Lgr5+ Intestinal Cells
5
Single-Cell RNA-Seq of Lgr5+ Intestinal Cells
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Lgr5-eGFP-IRES-CreER mice were treated with adenovirus in vivo and then 26h post-treatment, the proximal jejunum was harvested to generate a single cell suspension and FACS isolated using the endogenous GFP signal, as above. The sorted cellular suspensions were loaded on a GemCode Single Cell Instrument (10x Genomics, Pleasanton, CA) to generate single-cell GEMs. Approximately ~1200–2800 cells were loaded per channel. Two technical replicates were generated per sorted cell suspension. Single-cell RNA-Seq libraries were prepared using GemCode Single Cell 3′ Gel Bead and Library Kit (now sold as P/N 120230, 120231, 120232, 10x Genomics) as per Zheng et. al29 (link). Sequencing libraries were loaded at 2.1pM on an Illumina NextSeq500 with 2 × 75 paired-end kits using the following read length: 98bp Read1, 14bp I7 Index, 8bp I5 Index and 5bp Read2. Note that these libraries were generated before the official launch of GemCode Single Cell 3′ Gel Bead and Library Kit. Thus 5bp UMI was used (the official GemCode Single Cell 3′ Gel Bead contains 10bp UMI.).
6
Single-cell RNA-seq of nutlin-3 treated NGP cells
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Single cell RNA-seq on the Chromium system (10X Genomics) was performed for nutlin-3 (SI-GA-8E index) and vehicle (SI-GA-8D index) treated NGP cells using the GemCode Single Cell 3' Gel Bead and Library Kit (V2 chemistry, 10X Genomics, PN-120237, PN-120236, PN-120262) according to manufacturer's instructions with minor modifications. Cells were centrifuged at 4 °C at 400 g and resuspended in PBS + 0.04 % BSA to yield an estimated concentration of 1000 cells/µl. 3.5 µl of the cell suspension was used to obtain a cell recovery of about 2000 cells per sample. Per sample, 2.5 µl of an 1/10 dilution of ERCC spikes was added to the mastermix. After library preparation, the quality of the RNA libraries was confirmed on the Bioanalyzer.
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