NGS Services

Single Cell Gene Expression Analysis

While single cell sequencing can provide valuable insights into the complexity and heterogeneity of the cell types in the context of a tissue or tumor, there are challenges that exist that one needs to consider to be able to extract meaningful data from a single cell experiment. Key challenges during the workflow starts with preparation of single cell suspensions with good cell viability, efficiently capturing the diverse cell types in a mix via the appropriate cell capture method, and additional complexities with the quality of the data generated in a single cell experiment. Due to the tremendous utility of single cell gene expression in both basic as well as translational research, several approaches are now available for optimal isolating & scaling single cells isolation, preparing sequencing ready libraries that capture the maximal transcript diversity in a given cell, and analysis strategies to generate meaningful information from the data. At MedGenome, we have adopted several of the commercially available platforms for single cell gene expression analysis including the 10x Genomics Chromium platform for single cell gene expression and the SMART-Seq chemistries by Takara. As part of our service workflow we inspect the cell suspensions for viability and assess the quality of the sample prior to proceeding with the library preparation. We follow the manufacturer’s recommended guidelines for our quality control in every step of the process and provide customers with accurate QC reports.

Highlights of our services are:

  • Support with experimental design and selection of appropriate single cell workflow (dependent on cell types and number of cells)
  • Process single cell samples for a wide range of sample types (see table below)
  • Process single cell sequencing libraries from live and fixed tissues (methanol fixed only tested internally)
  • Provide end to end solution in library preparation, sequencing and bioinformatics analysis
  • Provide advanced bioinformatics analysis and data interpretation interpretation of data with advanced bioinformatics analysis solutions and data curation services

MedGenome’s Single-cell gene expression solutions workflow

Figure A: Workflow of single-cell sequencing services at MedGenome:

Depending on the type of sample, storage conditions of the sample (which effects the viability of the cells and quality of RNA obtained), and finally the number of cells available, a suitable workflow for library preparation is selected. In general, for fresh frozen tissues, upon optimization of tissue dissociation and freeze thaw conditions by the end user, frozen down cell suspensions are taken in for further processing through either the 10x Genomics Chromium gene expression solution or the Takara SMART-Seq workflow. In addition to fresh frozen, methanol fixed cells can also be processed using the chromium gene expression workflow. For samples with less than a few thousand viable cells as input (due to the final recovery of loss of 30% of cells in the processsing of cells), we recommend dissociating cells and sorting into plates and can perform the SMART-seq workflow. In addition to the low numbers of cells, if the samples include cell-types that are not pliable to freeze thaw, or have sizes over 40 μM, we recommend proceeding with SMART-seq.

Table A shows the sample requirements for single cell gene expression analysis

Sample type Number of viable cells Library prep used
Dissociated cells from frozen/ methanol fixed tissues, cell lines > 3000 cells( 80-90% minimum viability) 10x Genomics Chromium gene expression analysis
Dissociated cells from frozen tissue/cell lines < 3000 cells Takara SMART-Seq v4/ Takara SMART-Seq stranded (non-coding and stranded information)

Table B shows the cell types that have been processed at MedGenome and Summary of the data that was generated

Cell types Fresh frozen/ methanol fixed Cell viability Number of genes detected
PBMCs- 10X Data Not reported Not reported 1,297
PBMC – Source 1 Fresh frozen 99% 1,539
PBMC – Source 2 Fresh 87% 889
PBMC – Source 3 Fresh Frozen 89% 733
Primary hepatocytes Fresh frozen 80% 6,380
Mouse thymus dissociated tissue Fresh frozen 60% 851
Primary colon cells Fresh frozen 92% 4,127
Cancer cell lines treated with antibodies Methanol fixed Tumor cells: 92%
T cells: 57%
(before fixation)
T Cells – Source 1 Fresh 95% 2,850
T Cells – Source 2 Methanol fixed 95% 2,841

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