CSHL Single Cell Biology Core

To maximize the chance of a successful experiment please bring 100,000 cells or more for each sample of high viability cells. This ensures there are enough cells to make it through any pre run washes and buffer exchanges, and to accurately count and load the sample. Smaller cell numbers might still be possible, but you will need to consult with Core staff about your specific project. For scATAC, 100,000 or more cells are necessary to ensure that there are enough cells after the nuclei have been made.

Local Users: Local users can deliver samples to our Woodbury address in person as a single cell suspension by noon on the day of the scheduled experiment in .04%BSA in PBS for scRNA-seq or scATAC-seq. Shipping Samples: All shipped samples must include a copy of the agreed upon quote for the desired services and a list of included samples. scRNA-seq Single cell suspension must be shipped in a cryopreservation media in a cryovial on dry ice and will be stored in liquid nitrogen until they can be processed. snRNAseq Can be submitted as frozen tissue punches on dry ice If possible submit in a cryotube to easily allow us to store your samples on either dry ice or in liquid nitrogen until the run scATACseq Can be submitted as a cryopreserved single cell suspension or as a frozen tissue punch as the standard protocol involves making nuclei.

You have several options to sequence your 10x libraries. Most people choose to sequence their 10x libraries with the CSHL NGS core. If you choose to sequence your libraries with our NGS core we can submit them immediately after library preparation is complete. scRNAseq / snRNAseq / Immune Profiling Illumina NextSeq 2000 P2 run (suitable for sequencing up to ~10,000 cells to 40,000 reads/cell) or P3 flowcell (suitable for sequencing up to ~20,000 cells to 40,000 reads/cell) Read 1 = 28 bp (26 bp for 5’ chemistry), Read 2 = 90 bp and 10 bp i5 and i7 index reads Some applications may require longer reads - contact us for more information scATACseq Illumina NextSeq 500 High Output run Read 1 = 34 bp, Read 2 = 34 bp, a 16 bp i5 read of the cell barcode, and an 8 bp i7 index read For projects that require a large amount of sequencing some users choose to send their 10x libraries to other core facilities that have an illumina NovaSeq. We are also able to help in submitting libraries for long read sequencing (such as PacBio or Oxford Nanopore) with the CSHL NGS facility.

The general workflow for 10x Genomics scRNAseq (both 3’ and 5’ chemistries) begins with the cell suspension (either provided by the user or the result of upstream sample processing). Users can abort their run at any point before the Chromium instrument is loaded. After the instrument has been loaded the 10x run can no longer be aborted, but the user can still abort the illumina sequencing up until the library has been submitted to the NGS core for sequencing.

The workflow for 10x Genomics scATACseq can begin with either a cell suspension or a frozen tissue punch. Users can abort their run at any point before the tagmentation/ Chromium loading step. After the instrument has been loaded the 10x run can no longer be aborted, but the user can still abort the illumina sequencing up until the library has been submitted to the NGS core for sequencing.

Formula for cell recovery: Doublets: With microfluidics, the more concentrated the cell suspension, the higher the chances that a given droplet contains more than one cell. 0.9% per 1,000 captured cells (eg. expect 4.5% doublets in a 5,000 cell experiment) Sequencing considerations: Sequencing is purchased on a per-lane basis. Thus, you should plan your targeted number of cells based on the incurred sequencing costs. Typical depth: 40,000 reads per cell NextSeq 2000 P2 lane: 500M reads

In our experience, samples that are expected to yield < 1,000 cells after microfluidic capture are not worth running. If you have a particularly rare cell type or tiny input sample, consider a FACS-based single cell method instead.

We do not offer unassisted use of the sorter. All sorts must be performed with the assistance of Core staff.

The Core’s Sony SH800 can sort up to 6 colors. FL1: 425-475nm , FL2: 500-550nm, FL3: 570-630nm, FL4: 650-680nm, FL5: 690-750nm, and FL6: 755-815nm. For more information please see Sony SH800 Color Picker Guide

The Core keeps stock of DAPI, 7-AAD, NucBlue live, Propidium Iodide, and Acridine Orange on hand.

Information on how to access your data will be sent in the data delivery document when your data is ready. If you need further help accessing your data please email SingleCellBio@cshl.edu.

Yes. Custom references will be built using cellranger mkref. Genomes can be found on a database such as the Ensembl, RefSeq, or UCSC. In addition, a FASTA and GTF file need to be provided, which can also be found in a database such as Ensembl. Custom modifications and annotations are also welcome for FASTA and GTF files. Please contact a member of the core via email if you have questions or concerns.

Check the aggregation.csv or aggr.csv. Disregarding the header, the order of the sample names will correspond to the '-1', '-2'... suffix. Another solution would be to sum the suffices and see which sample matches the estimated number of cells in the web_summary.html. For more methods of associating the '-1', '-2', ... tags check out this biostar thread. You may also be interested in learning more about CellRanger's aggr function here.