Staff

Peter A. Sims, Faculty Director

Erin C. Bush, Director

Michael Finlayson, Bioinformatics Analyst

Izabela Krupska, Staff Associate

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 services using two scRNA-Seq platforms and an scATAC-Seq platform: the 10x Genomics Chromium 3' Solution and pooled scRNA-Seq in 96-well plates, and the 10x Genomics Chromium ATAC Solution.

The 10x Genomics Chromium Single Cell 3' Solution is a high-throughput, scalable technology that encapsulates thousands of individual cells 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 also prepare libraries for CITE-Seq, feature barcoding, CRISPR, or cell hashing. The resulting pooled, 3'-end libraries are typically sequenced on a NovaSeq 6000. Users must deliver live cells in suspension to the Core.

For scRNA-Seq in 96-well plates, users sort individual cells into the wells of a 96-well plate containing lysis buffer (provided by the Core). This step is performed at the user's convenience. After sorting, plates are spun down, frozen, and delivered to the Core where they are processed with our automated liquid handling robot. Template-switching reverse transcription is performed with adapter-linked oligo(dT) primers containing both cell- and molecule-specific barcodes, and cDNAs are pooled for PCR and library construction. Pooled, 3'-end sequencing libraries are then sequenced on an Illumina NextSeq 500/550. At least two plates are required per project.

The 10x Genomics Chromium Single Cell ATAC Solution 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 two methods provide different levels of throughput and flexible options. All first-time users are required to schedule an in-person meeting to discuss projects, please contact genome@columbia.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 subject to change. External users are welcome, but should request a quote.

10x Genomics Chromium 3' Single Cell RNA-Seq microfluidic cell processing, library preparation, and sequencing (~5,000 cells, ~350M reads): $2,738*

*CITE-Seq, cell hashing, and CRISPR barcoding library prep included for an additional fee, please inquire.

10x Genomics Chromium Single Cell ATAC-Seq microfluidic cell processing, library preparation, and sequencing (~5000 nuclei, ~130M reads): $3,104

Pooled 3'-End scRNA-Seq in 96-well plates, library preparation, and sequencing (~96 cells per plate): $613

96-well plate with reagents for Pooled 3'-End scRNA-Seq (for users to test or bank samples; library preparation and sequencing not included): $98

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 data processing (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:

10X Single Cell Gene Expression and 10X Single Cell ATAC-Seq

-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.

Plate-based Single Cell Gene Expression

-The standard data processing performed by our Core's pipeline

-Alignment of fastq files to a standard reference, a quality assessment, and the generation of count matrices for each sample individually. 

Tier 1, $250 per request*:

Available for 10X Single Cell Gene Expression and Plate-based Single Cell Gene Expression experiments

Includes:

-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, $500 per request:

Available for 10X Single Cell Gene Expression and Plate-based Single Cell Gene Expression experiments

Includes:

-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.

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 our web-based form .

For experiments using 10x Genomics Chromium 3' Solution, researchers must coordinate the delivery of a dissociated suspension of live cells. Cell preparation and dissociation is at the discretion of the researcher. We will perform a QC on the cells once they arrive, and the researcher can then give the go-ahead to move forward with the experiment.

For plate-based scRNA-Seq, researchers must submit 96-well plates containing the frozen lysates of individual cells sorted one at-a-time into each well. Researchers must use 96-well plates and reagents provided by the Core. Plates can be ordered and stored in advance.

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

Funding

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

PrecisionMedicine

 

IrvingInstitute

 

HICCC

 

ColumbiaMed