Parse Biosciences Single Cell Services

The Parse Evercode Whole Transcriptome assay is an approach to single cell RNA sequencing that doesn’t require an expensive, specialized instrument. Adapted from a method called Split Pool Ligation-based Transcriptome sequencing (SPLiT-seq), the Parse Evercode workflow employs a process called combinatorial barcoding, in which pooled cell suspensions are distributed throughout four different 96-well barcoding plates in sequence, wherein a unique oligonucleotide barcode is added to transcripts inside the intact cell. Unlike 10X Genomics, which achieves single cell resolution by partitioning individual cells inside of oil droplets, the ability to identify single cells in the Parse Evercode workflow is derived from the low statistical likelihood that any two cells will take the exact same path through the multiple rounds of barcoding. Because of this pooling-based approach, the final “sublibraries” generated for your samples will contain cells from every sample in your submission (which receive their sample-identifying barcodes in the first round of barcoding for de-multiplexing the data).

Parse Evercode kits are currently available in three sizes, allowing for the capture of approximately 10,000; 100,000; or 1,000,000 total cells or nuclei from up to 12, 48, or 96 samples, respectively. Please note that the input requires fixation of cells or nuclei using a kit sold by Parse Biosciences. In the Gene Expression Center, user-provided fixed cell suspensions will be used to generate Illumina-compatible library preps. These libraries will be transferred to the UWBC DNA Sequencing Facility for sequencing on the NovaSeq followed by optional analysis using the Parse analysis pipeline by the UWBC Bioinformatics Resource Core.

In addition to the Whole Transcriptome assay, Parse Biosciences also offers the Parse Evercode TCR assay for single cell immune repertoire profiling. Please e-mail to schedule a meeting to discuss your proposed experiment and timeline!

From the Parse Evercode WT v2.0 User Manual

Single Cell Suspension Submission Guidelines

  • Perform fixation with the Parse Biosciences Fixation Kit and follow the manufacturer’s protocol. The recommended input for fixation is between 100,000 and 4,000,000 cells/nuclei. Starting suspensions prior to fixation should have >70% viability and minimal cell aggregation/debris.
  • Please provide data on the starting cell viability and input cell/nuclei number along with any images you may have of the cell suspension on a cell counter/hemocytometer slide.
  • Fixed samples can be stored at -80C for up to 6 months prior to submission.
  • Parse Biosciences recommends a minimum of 20,000 reads per cell for clustering cells. For prioritizing transcript detection, a depth of 50,000 – 70,000 reads per cell may be more appropriate.
  • Cell concentration, viability, and singlet frequency is measured in the GEC using the Luna-FX7 Automated Cell Counter, which can do fluorescence-based cell counting with the nuclear stain AOPI.  We are happy to schedule “mock measurements” using the Luna to evaluate test samples prior to your real submission to ensure that maximum sample prep quality is achieved. Please be aware that a large component of this cell assessment process is qualitative with pass/fail thresholds based on human interpretation. We recommend you document (with images, if possible) the condition of the cells at the time of fixation. We cannot guarantee a successful experiment based on these cell counts alone and strongly encourage you to plan a small pilot experiment (e.g. with the 10,000 cell Mini Kit) first if your ultimate goal is a much larger submission.

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All data and services provided by the University of Wisconsin Biotechnology Center Gene Expression Center are intended for research purposes only. Services are not intended nor certified for diagnostic or clinical use.