microbiome block

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