Single-Cell Sequencing

To initiate a single-cell sequencing project, please contact us. Project consultation is provided free-of-charge. Consultation with the core prior to starting a single-cell sequencing experiment is highly recommended to ensure accomplishment of project goals.

Single-cell sequencing services can be requested through NUcore.

For all single-cell sequencing services, please make a sample submission appointment with us in advance.

• High-throughput single-cell sequencing platforms (10x Genomics, Illumina, and Parse): Users prepare fresh, frozen, or fixed cells (or nuclei) and submit them to NUSeq Core for sample QC and library construction.
• SMART-Seq platform: Users collect cell(s) in 0.2mL or 1.5mL tube in in 5µL of 1x PBS buffer and store at -80°C until sample submission to NUSeq Core.

10x Genomics Chromium Libraries

RNA-Seq Libraries: read length 28-10-10-90 bp — read 1 of 28 bp (cell barcode and UMI), i7 of 10 bp (sample index), i5 of 10 bp (sample index), and read 2 of 90 bp (transcript insert). Sequencing depth >20,000 reads per cell

ATAC-Seq Libraries: 50-8-16-50 bp — read 1 of 50 bp (transposed DNA), i7 of 8 bp (sample index), i5 of 16 bp (10x barcode) and read 2 of 50 bp (transposed DNA). Sequencing depth >25,000 reads per cell

Illumina Single Cell 3’ RNA Prep Libraries: 45-10-10-72 bp — read 1 of 45 bp (containing cell barcode), i7 of 10 bp (sample index), i5 of 10 bp (sample index), and read 2 of 72 bp (transcript insert, including intrinsic molecular identifiers or IMIs). Sequencing depth > 20,000 reads per cell

Parse Biosciences Libraries: 74-10-10-86 bp — read 1 of 74 bp (transcript insert), i7 of 10 bp (UDI), i5 of 10 bp (UDI), and read 2 of 86 bp (barcodes). Sequencing depth > 20,000 reads per cell

Manual Low-Throughput scRNA/scDNA-Seq: RNA-Seq — paired-end 50 bp reads at desired sequencing depth (e.g., 20-25 million reads per mammalian sample). DNA-Seq — Paired-end 150 bp reads often used at desired sequencing depth (e.g., 30x coverage for single-cell whole genome sequencing)

Data analysis is provided upon request.

From sample submission to library preparation it typically takes 2 weeks. It takes another 2-3 weeks for sample to be sequenced followed by up to 2 weeks for raw sequence demultiplexing and read alignment.

For cell, we recommend the cell viability of >70%. For nucleus, please consult us to ensure the sample quality is good for 10x Genomics platform.

Yes. This is achievable. Please consult the core for more specifics.

Yes. This is achievable. Please consult the core for more specifics.

Please contact NUSeq (NUSeq@northwestern.edu) and we will help you through the whole workflow from sample preparation to library construction to data analysis.

There are four steps in the workflow. First, cells are encapsulated into droplets along with barcoded hydrogel beads using an emulsification process, which is achieved with a vortexing process. Next, each cell is lysed in its droplet by heat-activated lysis reagents. This is followed by the capture of released mRNA molecules by bead-bound barcoded poly(T) oligonucleotides. The captured mRNA is then reverse transcribed into cDNA. The last step is preparation of sequencing libraries from the barcoded cDNA. The original method (called PIP-seq) is described in detail in Nature Biotechnology.

There are four kit sizes available: T2, T10, T20, and T100, designed for profiling 2K, 10K, 20K, and 100K target cells per sample, respectively. Among these, the T2 and T10 kits are designed for smaller-scale projects. T2 is especially suited for pilot, organoid or stem cell studies, preliminary data collection for grant applications, and cell sorting optimization.

Human: Brain, Retina, Lung, PBMCs, Bone marrow, Adipocytes, Ovary, Testes, Umbilical cord, Lung organoid, Cerebral organoid, Epithelial cells, and mesenchymal cells.

Mouse: Brain, Retina, Inner ear hair cells, Heart, Mammary tissue, Liver, Intestine, PBMCs, Bone marrow, Plasma, Erythrocytes, Bladder, Embryonic tissue, Cerebral organoids, Xenografts, Brain tumor, and Neutrophils.

Pig (liver and spleen), Zebrafish, Killifish, Xenopus, Planarians, Skate, Tunicates, Sea anemone, Taxoplasma (a single-celled parasite), Yeast, Maize, and Bovine (ovary).

Based on Illumina, the multiplet rate is less than 5%.

Yes. 

Please refer to the Illumina Single Cell Prep training packet, which breaks down the best practices for preparing your samples for the workflow.

Cells up to 60 µm in diameter have been successfully captured, although the exact upper limit has not yet been fully established. For cells ≥ 60 µm, Illumina recommends reducing cell input number to improve capture efficiency and minimize background noise. For cells > 80 µm, Illumina advises isolating nuclei and using them as input instead of whole cells.

Each platform has its own unique characteristics in terms of cell recovery, sensitivity in detecting genes and transcripts, cell population identification, and differential gene expression profiling. Currently available benchmark data (e.g., Elz et al., 2025), while still limited, shows that PIP-seq is still behind the latest 10x Genomics chemistries in cell recovery and gene/transcript detection, but compares well with Parse.

From sample submission to library preparation it typically takes 3 weeks. It takes another 2-3 weeks for sample to be sequenced followed by up to 2 weeks for raw sequence demultiplexing and read alignment.

For Parse Bioscience platform, all fixed samples should be submitted to us all at once. For 100,000 cell and 1,000,000 cell kit, users can submit up to 48 and 96 samples, respectively.

For cell, we recommend using 1 million cells with cell viability of >70% prior to fixation. For nucleus, please consult us to ensure the sample quality is good.

Yes. This is achievable. Please consult the core for more specifics.

Please contact NUSeq (NUSeq@northwestern.edu) and we will help you through the whole workflow from sample preparation to library construction to data analysis.

For sorted cell, please provide us 2-4 individual samples for protocol optimization. Please collect cell by sorter or manual pipetting in 0.2 mL tube with up to 3 µL of 1X PBS Buffer. Please store cells at -80°C until sample submission to NUSeq Core.

Please contact NUSeq (NUSeq@northwestern.edu) and we will help you through the whole workflow from sample preparation to library construction to data analysis.