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Single cell surface protein profiling-10XGenomics MRL

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Overview

Single cell surface protein profiling: This assay captures cell surface protein signatures. Cell surface proteins can be labeled using a specific antibody conjugated to a Feature Barcode oligonucleotide. This assay uses a Gel Bead-in-Emulsion (GEMs) concept to capture individual cell signals. In this workflow, two libraries are constructed that include a 3’ gene expression library and one Feature barcode library. RAW sequencing data is analyzed using the Cell Ranger pipeline and visualized through Loupe browser tools.

Technology

We use the following 10X Genomics kits and platforms for sequencing library preparation and sequencing.

  • Chromium Next GEM Single cell 3’ Kit v3.1
  • 3’ Feature barcode kit, 16 rxn
  • Dual Index Kit TT Set A 96rxn
  • Chromium Next GEM Chip G Single cell kit, 16 rxn
  • Library constructions kit, 16 rxn
  • NovaSeq6k-S4

Key Features

  • Reveal cell surface protein and gene expression from the same cell
  • Understand diverse CRISPR-mediated perturbations at single cell resolution
  • Single cell gene expression analysis
  • High resolution cell type characterization
  • Precise cell surface gene expression
  • Cell type composition

Third-Party Resources

Cell Ranger and Loupe Browser | Single cell data analysis and data visualization tools

The Loupe Browser is a desktop application that provides interactive visualization functionality to analyze data from different 10x Genomics solutions. Cell Ranger and Loupe Browser

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Single cell Multiomics-10XGenomics MRL

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Overview

Simultaneous capture of gene expression and open chromatin signatures from the same cell with Chromium Single Cell Multiome ATAC + Gene Expression allows multimodality and high resolution functional characterization of genomics and transcriptional states. This assay uses a Gel Bead-in-Emulsion (GEMs) concept to capture individual cell signals. In this workflow, two libraries are constructed that include a 3’ gene expression library and one ATAC-seq library. About 10K individual cells are input in each library and 30-40K paired sequencing reads are generated on gene expression library and 50K reads on ATAC-seq library. These libraries are sequenced on NovaSeq6k platforms to generate 300-600 Million sequences per sample. RAW sequencing data is analyzed using the Cell Ranger pipeline and visualized through Loupe browser tools.

Technology

We use the following 10X Genomics kits and platforms for sequencing library preparation and sequencing.

  • Chromium Next GEM Single cell 3’ Kit v3.1
  • ATAC-seq kit
  • Dual Index Kit TT Set A 96rxn
  • Chromium Next GEM Chip G Single cell kit, 16 rxn
  • NovaSeq6k-S4

Key Features

  • Single cell gene expression analysis
  • Chromatin state signature
  • High resolution cell type and state characterization
  • Precise gene expression regulation analysis
  • Cell type composition

Third-Party Resources

Cell Ranger and Loupe Browser | Single cell data analysis and data visualization tools

The Loupe Browser is a desktop application that provides interactive visualization functionality to analyze data from different 10x Genomics solutions. Cell Ranger and Loupe Browser

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Fixed RNA Single cell RNA sequencing-10XGenomics MRL

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Overview

The Single Cell Fixed RNA Profiling (FRP) workflow measures RNA levels in samples (single cells or nuclei) fixed with formaldehyde, using probes targeting the whole transcriptome. After whole transcriptome probe panels are added to the sample, each probe pair hybridizes to its target gene and is then ligated. Libraries are generated from the barcoded probes and sequenced. The final sequencing library structure is similar to Gene Expression solutions for fresh frozen tissue, but the synthetic probe DNA is sequenced rather than the cDNA of a captured transcript. Our Core offers this service. Investigators will need to prepare and fix their cells following 10X instructions for this assay.

Technology

We use the following commercially available kits and platforms for sequencing library preparation and sequencing.

  • Chromium Fixed RNA kit, Human/Mouse Transcriptome, 4 rxn
  • Chromium Next GEM Single Cell Fixed RNA Sample Prep Kit, 16 rxn
  • Chromium Next GEM Chip Q Single Cell Kit, 48 rxn
  • Dual Index Kit TS Set A, 96 rxn

Key Features

  • Gene expression analysis on fixed cells or nuclei
  • Process cells fixed with formaldehyde-fixed samples
  • Increased sample throughput in a single GEM well with multiplexing

Third-Party Resources

Cell Ranger and Loupe Browser | Single cell data analysis and data visualization tools

The Loupe Browser is a desktop application that provides interactive visualization functionality to analyze data from different 10x Genomics solutions. Cell Ranger and Loupe Browser

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5’ Single cell RNA sequencing-10XGenomics MRL

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Overview

Chromium Single Cell Immune Profiling allows analysis of full-length, paired B-cell or T-cell receptor (BCR/TCR) sequences, surface protein expression, antigen specificity, and 5’ gene expression, all from a single cell. Scale up with a high-throughput solution, or focus on key genes with targeted gene expression panels. This assay uses a Gel Bead-in-Emulsion (GEMs) concept to capture individual cell signals. In this workflow, three libraries are constructed that include a 5’ gene expression library, one TCR library and one BCR library. About 10K individual cells are input in each library and 30-40K paired sequencing reads are generated on gene expression library and 5-10k reads on TCR and BCR library. These libraries are sequenced on NovaSeq6k platforms to generate 300-500 Million sequences per sample. RAW sequencing data is analyzed using Cell Ranger pipeline and visualized through Loupe browser tools.

Technology

We use the following 10X Genomics kits and platforms for sequencing library preparation and sequencing.

  • Chromium Next GEM Single cell 5’ Kit v2
  • Dual Index Kit TT Set A 96rxn
  • Chromium Next GEM Chip K Single cell kit, 16 rxn
  • Chromium Single Cell Mouse/Human BCR amplification Kit, 16rxn
  • Chromium Single Cell Mouse/Human TCR amplification Kit, 16rxn
  • NovaSeq6k-S4

Key Features

  • Immune Cell Profiling assay
  • 5’ Single cell gene expression analysis
  • High resolution immune functional characterization
  • Precise pathway analysis
  • Cell type compositionTHIRD-PARTY RESOURCES (multiple)

Third-Party Resources

Cell Ranger and Loupe Browser | Single cell data analysis and data visualization tools

The Loupe Browser is a desktop application that provides interactive visualization functionality to analyze data from different 10x Genomics solutions. Cell Ranger and Loupe Browser

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3’ Single cell RNA sequencing-10XGenomics MRL

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Overview

An organ/tissue consists of a heterogeneous population of cells. Each individual cell performs an intricate role to the ensemble. Because of this complexity, gene expression research in organisms is often limited by traditional bulk RNA-seq methods as the obtained signal cannot be tracked to the original cell. Our core offers Chromium Single Cell Gene Expression assay which provides single cell transcriptome 3′ gene expression capabilities to profile tens of thousands of single cells in parallel. This assay uses a Gel Bead-in-Emulsion (GEMs) concept to capture individual cell signals. It uses a polydT primer for reverse transcription. In this assay the polydT sequence is located on the gel bead oligo. After amplifying the cDNA, molecules are randomly fragmented under conditions that favor 300-400 bp length fragments. Downstream of fragmentation, only transcripts containing both (1) a 10x Barcode AND (2) an Illumina Read 2 adaptor, which is ligated on to the cDNA after fragmentation, are amplified during the Sample Index PCR. This results in final 10x libraries that either represent the 3′ end of the transcript (as the 10x Barcode is adjacent to the polyA tail on the 3′ end of the transcript). About 10K individual cells are input in each library and 30-40K paired sequencing reads are generated per cell to robustly quantify the gene expression. These libraries are sequenced on NextSeq550 or NovaSeq6k platforms to generate 300-500 Million sequences per sample. RAW sequencing data is analyzed using the Cell Ranger pipeline and visualized through Loupe browser tools.

Technology

We use the following 10X Genomics kits and platforms for sequencing library preparation and sequencing.

  • Chromium Next GEM Single cell 3’ Kit v3.1
  • Dual Index Kit TT Set A 96rxn
  • Chromium Next GEM Chip G Single cell kit, 16 rxn
  • NovaSeq6k-S4

Key Features

  • Single cell gene expression analysis
  • High resolution functional characterization
  • Precise pathway analysis
  • Cell type composition

Third-Party Resources

Cell Ranger and Loupe Browser | Single cell data analysis and data visualization tools

The Loupe Browser is a desktop application that provides interactive visualization functionality to analyze data from different 10x Genomics solutions. Cell Ranger and Loupe Browser

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Metatranscriptomics Illumina MRL

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Overview

Metatranscriptome sequencing delivers the whole expression profile by randomly sequencing mRNAs and tracks the dynamics of gene expression patterns in microbial communities using next-generation sequencing (NGS). This assay generates data to study activity of a microbiome community in the context of the niche or any aspects of health and disease. Typically, microbiome assay uses 16S rRNA gene sequencing to look at overall bacterial diversity or shotgun metagenomics to determine the functional potential of microbes at the strain level. To complement these DNA-based methods, RNA sequencing, or meta- transcriptomics, provides direct information about active gene expression to understand the metabolic profile of the microbiome. This assay requires a high quality RNA as input. This workflow involves removal of ribosomal rRNAs species prior to sequencing library preparation which enables researchers to focus on analyzing high-value, informative portions of the transcriptome. RNAseq libraries are sequenced on NextSeq550 or NovaSeq6k flow cells to generate 50-100 Million reads per sample.

Technology

We use the following commercially available kits and platforms for sequencing library preparation and sequencing.

  • KAPA Biosystem RNA library prep kit
  • Zymo Research RNAseq library prep kit
  • NextSeq550-High Output Flow cell
  • NovaSeq6k-S4

Key Features

  • Gene expression analysis
  • Pathway analysis
  • Metabolite prediction
  • Antibiotic potential prediction
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Shotgun-Metagenomics Illumina MRL

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Overview

The Shotgun-Metagenomics is an assay used to study microbial communities using the whole genome sequencing approach. DNA extracted from all the cells in a community is subsequently sheared into tiny fragments that are independently sequenced. This results in DNA sequences that align to various genomic locations for the myriad genomes present in the sample, including non-microbes. Some of these reads will be sampled from taxonomically informative genomic loci (e.g., 16S), and others will be sampled from coding sequences that provide insight into the biological functions encoded in the genome. As a result, metagenomic data provides the opportunity to simultaneously explore both structure and potential functions of a microbial community: who is there and what they are capable of doing. Sequencing depth range 100-200 Million reads per sample.

Technology

We use the following commercially available kits and platforms for sequencing library preparation and sequencing.

  • KAPA Biosystem DNA library prep
  • Nextera DNA Flex from Illumina
  • NextSeq550-High Output Flow cell
  • NovaSeq6k-S4

Key Features

  • Whole genome data based characterization
  • Gene function prediction
  • Potential pathway prediction
  • Antibiotic potential prediction
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Pathogen-Genome Sequencing Nanopore MRL

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Overview

This assay uses Oxford nanopore technology to generate long-read sequencing data on microbial samples. This assay generates long sequencing reads for complete genome assembly. Assay requires about 500-1000ng high molecular weight genomic DNA as input. The greater overlap offered by long sequencing reads enhances genome assembly by providing longer continuous, unambiguously assembled sequences, resulting in fewer contigs. Accurately resolve structural variants and repeat regions, and characterize base modifications (e.g., methylation), fusion genes, and haplotype phasing, with Oxford nanopore long reads.

Technology

We use the following commercially available kits and platforms for sequencing library preparation and sequencing.

  • Nanopore Native Barcoding kit
  • R9.4.1/ R10 flow cell
  • MinION/Mk1c/GridION

Key Features

  • Long sequencing reads
  • Unambiguous identification of structural variants i.e. Insertions, Deletions, etc.
  • Accurate read alignments
  • No PCR bias
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Pathogen-Genome Sequencing on Illumina

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Overview

Whole-genome sequencing (WGS) is a comprehensive assay for analyzing the entire genome. This assay generates deep sequencing data on genomic DNA extracted from bacterial culture or clinical isolates. About 500-1000 ng of high complexity genomic DNA is required for sequencing library preparation using kits from KAPA biosystem, Illumina, etc. Genomic fragments of 300-500bp size are ligated with Illumina recommended adapters containing unique barcodes or Unique Molecular Identifiers (UMIs). These libraries can be sequenced on MiSeqDx, HiSeq or NextSeq platforms to achieve 70-100 fold sequencing coverage across the entire genome.

Technology

We use the following commercially available kits and platforms for sequencing library preparation and sequencing.

  • Illumina FLEX DNA/ KAPA Biosystem Library prep Kits
  • MiSeqDX 300 cycle Flow cell
  • NextSeq550 150 cycle Flow cell
  • NovaSeq 6K PE150 sequencing

Key Features

  • Full-length transcripts
  • Unambiguous identification of splice variants and fusion events
  • Accurate isoform quantification
  • No PCR bias
  • Detects base medications (epigenetic changes) alongside nucleotide sequence
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Native RNA Sequencing on Nanopore

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Overview

This assay allows complete quantification and characterization of native RNA or cDNA molecules present in a sample. A sequencing library is constructed on poly-A enriched RNA followed by sequencing on MinION/Mk1c platform from Oxford Nanopore.

Technology

We use the following commercially available kits and platforms for sequencing library preparation and sequencing.

  • Nanopore Native RNAseq library prep
  • MinION R9.4.1 sequencing
  • EPI2ME analysis pipeline

Key Features

  • Full-length transcripts
  • Unambiguous identification of splice variants and fusion events
  • Accurate isoform quantification
  • No PCR bias
  • Detects base medications (epigenetic changes) alongside nucleotide sequence