Staff

Peter A. Sims – Faculty Director

Erin C. Bush – Director

Izabela Krupska – Senior Staff Associate

Lucy Anisimov – Staff Associate

Kwasi Osae-Kwapong – Bioinformatician

Simoni Tiano – Bioinformatician

Joseph Mullen – Technician

Contact

genome@colubmia.edu

Overview

The Single Cell Analysis Core provides experimental and computational support for single-cell RNA-Seq (scRNA-Seq) and single-cell ATAC-Seq (scATAC-Seq). The Core was launched with generous support from the Columbia Precision Medicine Initiative, the Irving Institute for Clinical and Translational Research , the Herbert Irving Comprehensive Cancer Center , and the Department of Medicine . We are excited to provide multiple services using the 10x Genomics Platform: 3’ Single Cell Gene Expression, 5’ Single Cell Gene Expression (plus V/D/J sequencing), Single Cell ATAC, and Single Cell Multiome ATAC + Gene Expression.

The 10x Genomics Single Cell Gene Expression 3’ and 5’ workflows are both high-throughput, scalable technologies that encapsulate thousands of individual cells (or nuclei) in droplets for lysis and reverse transcription from up to 8 samples in parallel. The droplets contain primers for cell- and and molecule-specific barcoding of cDNA. We can prepare libraries for CITE-Seq/feature barcoding, CRISPR, or cell hashing. Using our 5’ workflow, we can profile the full length V(D)J regions from T-cell or B-cell receptors from human and mouse samples. The resulting libraries are typically sequenced on an Illumina NovaSeq 6000. Users must deliver live cells (or nuclei) in suspension to the Core.

 The 10x Genomics Single Cell ATAC workflow is a comprehensive, scalable approach to determine the regulatory landscape of chromatin in thousands of cells in a single sample. Nuclei are delivered in suspension to the Core, then transposed in bulk before being encapsulated in droplets for individual nuclei barcoding. The resulting libraries are typically sequenced on an Illumina NextSeq 500/550.

 The 10x Genomics Multiome ATAC + Gene Expression workflow simultaneously profiles the epigenomic landscape and gene expression in the same single nuclei for thousands of nuclei in parallel. As above, nuclei are transposed in bulk to add adapters to the ends of the DNA before being encapsulated in droplets. The droplets contain reagents and beads with a poly(dT) sequence that enables production of barcoded, full-length cDNA from poly-adenylated mRNA for gene expression (GEX) library and a Spacer sequence that enables barcode attachment to transposed DNA fragments for ATAC library. Resulting libraries are typically sequenced on an Illumina NovaSeq 6000 or a NextSeq 500/550.

 All first-time users are required to schedule an in-person meeting to discuss projects, please contact genome@colubmia.edu to schedule a meeting.

Instrumentation

In addition to the capital equipment assets in the Sulzberger Columbia Genome Center, the Single Cell Analysis Core maintains the following instruments:

10x Genomics Chromium Single Cell Controller

Beckman-Coulter Biomek 4000

ThermoFisher Countess II FL Automated Cell Counter

Miltenyi gentleMACS Octo Dissociator (available for self-service use, please contact genome@columbia.edu).

Standard Packages and Pricing*

Current prices are for Columbia users, are subsidized by institutional support, and are subject to change. External users are welcome, but should request a quote.

10x Genomics 3’ or 5’ Single Cell Gene Expression (GEX) microfluidic cell processing, library preparation, sequencing, and basic analysis* (default target ~5,000 cells, ~350M reads): $2,626

          Library prep for feature barcoding/CITE-Seq, cell multiplexing, CRISPR barcoding, and TCR/BCR: $50 each

          CellPlex Oligos: $85 each

10x Genomics Single Cell ATAC-Seq microfluidic cell processing, library preparation, sequencing, and basic analysis (default target ~5000 nuclei, ~130M reads): $3,255

10x Genomics Single Cell Multiome microfluidic cell processing, library preparation, sequencing, and basic analysis (default target ~5000 nuclei, ~350M reads for GEX libraries, ~130M reads for ATAC libraries): $5,374

Sample Submission

All first-time users are required to schedule an in-person meeting to discuss projects (genome@columbia.edu). Once the date and time is decided and agreed upon by the Core, sample and billing information can be submitted using the following procedure:

  1. Please fill in a submission form in iLab. This is for either a 10x request or an analytics request. You’ll need the service ID generated for the second form.
  2. Please fill in a form for internal tracking, this will ask for your iLab service ID from the previous step: Submission Form

Both forms are required. We cannot help with chartstring management, please discuss with your PI to make sure you have access to the chartstring you need.

 For Gene Expression experiments, researchers must coordinate the delivery of a dissociated suspension of live cells or nuclei (please email genome@columbia.edu to set this up). Cell preparation and dissociation is at the discretion of the researcher. We will perform QC on the cells/nuclei once they arrive, and the researcher can then give the go-ahead to move forward with the experiment. CellPlex oligos can be picked up a few days before your experiment, please coordinate with the Core. All labeling reactions are performed by the researcher.

 Additional library preparations (feature barcoding/cell multiplexing/CRISPR/TCR/BCR) must be discussed with the Core prior to submission, indicated in both your submission and internal tracking forms, and communicated to our staff associates upon loading the cells.

For scATAC-Seq or Multiome experiments, nuclei must be extracted from the cells and delivered in suspension. We will resuspend the nuclei in the correct buffer for tagmentation and perform QC, and the researcher can then give the go-ahead to move forward with the experiment.

Bioinformatics Packages

We recognize that scRNA-Seq presents numerous analytical challenges, and that computational techniques in this area are rapidly evolving. We are therefore pleased to provide bioinformatics support for processing, quality control, analysis, and interpretation of scRNA-Seq data generated by the core as an additional service as described below. In addition to basic analysis included in packages above (barcode demultiplexing, alignment, molecular counting), the Single Cell Analysis Core also provides state-of-the-art computational tools for identifying cellular subpopulations, differential expression, data visualization, and statistical analysis. Please contact  genome@columbia.edu to learn more and schedule a consult with our experts.

Free Tier, included in all Standard Packages:

-The standard output of 10X's Cell Ranger analysis software.

-Specifically, this means processing fastq files using "cellranger count" for each sample individually with default parameters.

-This produces an alignment of reads to a standard reference, a quality assessment, a count matrix, a clustering, and a differential expression analysis targeted at markers specific to individual clusters.

Tier 1, $250 per request*:

-Realignment with an altered genome/annotation

-Reanalysis/reprocessing with different versions of cellranger or different refrences

-Clustering and differential expression analysis with our Core's standard analysis routines (for 10X experiments, this refers to analyses beyond the standard "cellranger count" process

-Integration of multiple samples using cellranger or our Core's standard analysis routines 

Tier 2, Tier 1 flat fee + $75 per hour (quote provided before proceeding):

-Iterative clustering and differential expression with our core’s standard analysis

-Generating custom figures

-Trajectory analysis

-Comparison to a reference dataset

-Any analysis not included in Tier 1

*A request for analysis is defined as a request to perform one task repeatedly on a manageable number of samples that are part of one experiment, and the tier will be agreed upon by the researcher and the Core before beginning the analysis. A request can be made at any time, either before submitting the samples or after receiving data.

Analytics-only Tier, $50 per sample:

Required for all self-prepared libraries (10X or plate-based) processed using the Single Cell Analysis Core’s pipelines. All Free Tier analyses are included.

Funding

The Single Cell Analysis Core gratefully acknowledges the following organizations for their generous support:

PrecisionMedicine

 

IrvingInstitute

 

HICCC

 

ColumbiaMed