Single-cell RNA sequencing (sc-RNA seq) was performed using the 10x genomics chromium single cell controller. Briefly, tumor cell single cell suspensions were prepared as indicated above. Cells were resuspended in freezing media containing 90% AB serum and 10% DMSO and stored in liquid nitrogen until analysis. For sc-RNA seq analysis cells were thawed, washed and sorted for viable CD45+ cells using the BD FACSAria. Next, cells were droplet separated using Chromium™ Single Cell 3′ v2 Reagent Kit with the 10x genomics microfluidic system creating cDNA library with individual barcodes for individual cells. Barcoded cDNA transcripts from GBM patients were pooled and sequenced using the Ilumina HiSeq 4000 Sequencing System.
Single cell 3 reagent kit v2
Manufactured by 10x Genomics
Sourced in United States
The Single Cell 3' Reagent Kit v2 is a laboratory equipment product developed by 10x Genomics. The kit is designed to enable the analysis of individual cells, providing a tool for researchers to study gene expression patterns at the single-cell level.
Automatically generated - may contain errors
Lab products found in correlation
Chromium controller, by 10x Genomics (7 mentions)
Chromium system, by 10x Genomics (3 mentions)
Tc20 automated cell counter, by Bio-Rad (3 mentions)
Hiseq 2500, by Illumina (2 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (2 mentions)
Chromium controller instrument, by 10x Genomics (2 mentions)
Spriselect reagent kit, by Beckman Coulter (2 mentions)
Hiseq platform, by Illumina (2 mentions)
Chromium single cell 3 library gel bead kit v2, by 10x Genomics (2 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
Nextseq, by Illumina (2 mentions)
Chromium platform, by 10x Genomics (2 mentions)
Facsaria, by BD (1 mentions)
Hiseq 4000 sequencing system, by Illumina (1 mentions)
Nexseq 500, by Illumina (1 mentions)
Cell ranger platform, by 10x Genomics (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Gemcode, by 10x Genomics (1 mentions)
Tumor dissociation kit, by Miltenyi Biotec (1 mentions)
Hiseq x platform, by Illumina (1 mentions)
28 protocols using single cell 3 reagent kit v2
1
Single-cell RNA Sequencing of Tumor Cells
2
Single-cell RNA-seq of NP-specific CD4+ T cells
3
Single-cell RNA sequencing of 1,086 cells
4
Single-Cell RNA-seq of E14.5 Mouse Embryos
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E14.5 C57BL/6 WT embryos were used for droplet-based RNA-seq. Tissue dissociation was performed as previously described for microfluidic embryonic preparation. Cell suspension was loaded into a 10x Chromium Controller (10x Genomics) and processed with the Single Cell 3′ v2 reagent kit (10x Genomics) according to the manufacturer's protocol. Briefly, single cells were partitioned into gel beads in emulsion (GEMs) in a 10x Genomics GemCode instrument followed by cell lysis and barcoded reverse transcription of RNA, amplification, shearing and 5′ adaptor and sample index attachment. For the POA and CGE E14.5 libraries, 4978 and 2736 cells were recovered, respectively, after being sequenced on a HiSeq 4000 instrument (Illumina) at an expected depth of 70,000 reads per single cell. ‘Cell Ranger’ software (10x Genomics, version 3.0.2) was used for mapping reads to the mouse genome provided by the instrument manufacturer (10x Genomics, mm10 refdata v3.0.0) and for generating feature-barcode matrices.
5
Bulk and Single-cell RNA Sequencing of Prostate Cancer Tumor Models
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For bulk RNA sequencing of PC-3 and PC-42 tumors, total RNA was extracted by using a Maxwell RSC simplyRNA Tissue Kit (Promega), and the cDNA library was prepared with an Agilent SureSelect Strand Specific RNA Library Prep Kit (Agilent). Reads (150 bp, paired-end) were sequenced on Illumina NovaSeq (12 Gb per sample). Reads were separated by species of origin by using Human.GRCh38 and Mouse.mm10. For single-cell analysis, PC-3 and PC-42 tumors were dissociated by using a tumor dissociation kit (Miltenyi Biotec). After stromal cell isolation with a Mouse Cell Depletion Kit (Miltenyi Biotec), single-cell suspensions of mouse stromal cells (~5000 cells) were loaded into a 10x Chromium Controller (10x Genomics) with a Single Cell 3′ v2 reagent kit (10x Genomics). The libraries were sequenced on a HiSeq X platform (Illumina), and the sequenced reads were processed by using CellRanger software (10x Genomics, v3.0.2). Stromal data from PC-3 and PC-42 tumors were merged, and clustering by t-SNE was performed with Loupe Browser (10x Genomics, v5.1.0). RNA sequence data of KYK models were obtained from KAN Research Institute. RNA sequence data of TCGA were downloaded from UCSC Xena Data Hubs.
6
Single-cell RNA-seq Library Preparation
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According to the manufacturer’s protocol, the single-cell suspension was loaded into the Chromium single-cell controller (10X Genomics) for single-cell capturing and downstream library constructions with the Single Cell 3′v2 reagent kit (10x Genomics). Briefly, single cells were suspended and recovered, aiming for 6000 cells. Cells were partitioned into Gel Bead-In-EMulsions (GEMs) with barcoded gel beads. Following cell capture and lysis, complementary DNA was generated and amplified via a S1000TM Touch Thermal Cycler (Bio-Rad). Then, the sequencing libraries were constructed and loaded on an Illumina NovaSeq 6000 sequencer for sequencing.
7
Single-cell RNA-seq of Murine Bone Marrow
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BMCs were harvested from 16-week-old C57BL/6 mice. CD117+ cells were obtained with CD117 microbeads (Miltenyi Biotec) using the Lineage Cell Depletion Kit (mouse). The 10x Genomics Single Cell 3′ v2 Reagent Kit was used to prepare samples according to specific instructions. The cells were then counted, and approximately 6000 cells from every sample were loaded onto a 10x Genomics single-cell-A chip to construct the cDNA library in accordance with the instructions of the Single Cell 3′ Reagent Kit v3 after some modifications of PCR cycles based on the determined cDNA level (recommendations from 10X Genomics). Each sample was then combined, followed by normalization to 10 nM and dilution with an elution buffer containing 0.1% Tween 20 (Sigma) to 2 nM. Later, Novaseq 6000 was used for sample sequencing with the following parameters: Read 1-26 cycles, read 2-98 cycles, and index 1-8 cycles. Each sample was sequenced to the median depth of 50,000 reads/cell.
8
Single-cell RNA sequencing of PDX
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In order to perform scRNA-seq, the primary sample (BC159-T#3) was directly obtained from the operating room, and PDX was generated from this primary sample. Specimens were dissociated into single cells according to previously published protocols [11 (link), 35 (link)]. After resuspension in 1× phosphate-buffered saline, all single-cell suspensions were loaded into a 10x Chromium Controller (10x Genomics, Pleasanton, CA, USA), aiming for 7000 cells, with the Single Cell 3′ v2 reagent kit (10x Genomics), according to the manufacturer’s instructions. Following Gem capturing and lysis, cDNA was synthesized and amplified to construct sequencing libraries. The libraries were sequenced on the Illumina HiSeq 2500 platform, and sequenced reads were processed using the CellRanger toolkit (version 2.1.0). The human and mouse genomes were mapped to the GRCh38 human genome reference and mm10 mouse genome reference, respectively, using STAR [36 (link)].
9
Single-cell transcriptome profiling of lung cells
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mRNA from single cells sorted from lung into lysis plates was reverse transcribed to complementary DNA (cDNA) and amplified. Library preparation and sequencing were performed. Sequencing libraries for cDNA from single cells were prepared as per the Single Cell 3′ v2 Reagent Kits User Guide (10x Genomics, Pleasanton, CA, USA). Cellular suspensions were loaded on a Chromium Controller instrument (10x Genomics) to generate single-cell Gel Bead-In-EMulsions (GEMs). GEM-reverse transcription (RT) was performed in a Veriti 96-well thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). GEMs were collected and the cDNA was amplified and purified with SPRIselect Reagent Kit (Beckman Coulter, Brea, CA, USA). Indexed sequencing libraries were constructed using Chromium Single-Cell 3′ Library Kit for enzymatic fragmentation, end-repair, A-tailing, adapter ligation, ligation cleanup, sample index PCR, and PCR cleanup. The barcoded sequencing libraries were quantified by quantitative PCR using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems, Roche Holding AG, Basel, Switzerland). Sequencing libraries were loaded on a NextSeq500 with a custom sequencing setting (26bp for Read 1 and 98bp for Read 2) to obtain a sequencing depth of ~200K reads per cell.
10
Single-Cell Transcriptomic Analysis Protocol
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