The Flow Cytometry Facility also provides a Chromium 10X single cell service to the institute. Image Next GEM Technology The Chromium platform, powered by Next GEM technology, enables integrated analysis of single cells at massive scale. The key to this technology is the ability to generate tens of thousands of single cell partitions, each containing an identifying barcode for downstream analysis. Every Chromium solution starts with a high-diversity pool of Gel Beads each with a unique oligonucleotide barcode and sequence to capture molecules of interest. Within the Chromium instrument, barcoded Gel Beads are mixed with the cells or nuclei, enzymes and oil to create 'GEMs' (Gel Bead-in-emulsion) Each GEM droplet is an individual reaction where the Gel Beads are dissolved and molecules of interest are captured and barcoded. Once barcoded, all fragments from the same cell share the 10x barcode. The barcoded fragments are then pooled for downstream reactions to create short-read sequencer compatible libraries. After sequencing, bioinformatic tools use the unique barcodes to map sequencing reads back to their single cell of origin. Facility Information The 10x facility has a dedicated 10x technician to perform all stages in the preparation of the 10x libraries. Currently the facility offers Chromium Next GEM Single Cell 3' v3.1 reagents and chips to researchers but can advise, help order and perform full library preparation protocols for various 10x products. All 10x runs are performed on our in-house Chromium Controller instrument To book a 10x slot or to discuss further requirements please email FACS@igc.ed.ac.uk Please note all samples need to be received by 2pm on day of submission. FAQs Q: Who can use the services offered by the facility? A: Our facility is open to research groups both external and internal to the IGC. Q: What type of starting material can be used as input for a scRNA-seq experiment? A: Both cells and nuclei can be used as input, all starting material should be a clean suspension with high viability. For a provided single-cell or nuclei suspension we require the cell stock concentration (cells/µl) to be provided in order to load the chip accurately. Q. Do you provide a service for nuclei preparation from samples (tissue/cells)? A: No we do not provide this service. There are suggested protocols from 10x in our useful links section for your information. Q. Do you provide sorting facilities for samples? A: The 10x facility works closely with our FACS facility here at the IGC and can co-ordinate experiments so the 10x can be performed immediately after sorting of samples. Please email FACS@igc.ed.ac.uk for further information regarding using this facility. Q: How many cells or nuclei can you load in a scRNA-seq experiment? A: To have a targeted recovery of 10,000 cells/nuclei a maximum of 20,000 can be loaded onto the 3' v3.1 chip. Researchers have the flexibility to specify number of cells to be sequenced from 500 to 10,000 cells per samples (as stated in 10x protocol) but we recommend a targeted recovery of 5000 cells per sample as a minimum, loading a minimum of 10,000 cells onto the chip. Q: How long is the 10x library preparation process? A: A typical workflow can be carried out over 2-3 days from cells/nuclei to library for the 3’ experiment. Other kits may require additional steps but we aim to have a completed library ready for sequencing within 3 days. Q: Do you provide a service for spatial transcriptomics? A: No the service isn’t available here at the IGC. Q: Do you also sequence the libraries? A: Our facility doesn't provide any sequencing services for 10x libraries but can put you in contact with the Welcome Trust Clinical Research Facility (based at the Western General) who can provide Illumina sequencing information/pricing on request. We also co-ordinate with them to handle the sample submission process on your behalf. Q: Do you offer bioinformatics/data analysis support? A: No we do not provide this within the 10x facility but the IGC Bioinformatics Analysis Core can assist in any enquiries. IGC Bioinformatics Analysis Core Useful Links 10x Genomics Support Isolation of Nuclei for scRNAseq Sample Prep for cultured cell lines for scRNAseq Getting started sc3' Gene Expression Finding the 10x product for your experiment This article was published on 2024-09-23
