We support you in all steps of your scientific projects in high-throughput functional genomics: feasibility study, support for your grant requests, experimental design advices, sample management, training and use of equipments accessible by booking.
The core facility is equipped with a NextSeq 2000 high-throuput sequencer (Illumina).
For the applications below, we prepare and sequence your libraries, performing quality controls at each step.
We can also sequence your own libraries as long as they are compatible with Illumina technology.
The core facility does not realize nucleic acids extraction → See below for samples preparation and shippping recommandations upon applications.
You have a project requiring another application, we are at your disposal, do not hesitate to contact us → Contact us
RNAseq
The RNAs extraction as well as their quantitative and qualitative control are not provided by the platform. In order to obtain good quality RNAs, we recommend you to use extraction protocols based on columns with silica membrane or magnetic beads (Qiagen, Macherey Nagel, etc.) and no Trizol or phenol-chloroform.
The RNAs quality must be checked by high-resolution electrophoresis (Bioanalyzer, Tapestation, Labchip GX). The RIN (RNA Integrity Number) must be greater than or equal to 7.
However, there are specific protocols allowing for preparation of libraries from degraded RNAs from FFPE tissues.
Library preparation protocols depend on the amount of total RNA available as well as the type of RNA you want to study → Contact us
Sequencing options on NextSeq 2000
Flow cells can be used for single read (SE) or paired-end (PE) sequencing.
For example, P2 100bp flow cell can be used for SR 100bp or PE 2x50bp sequencing.
Cell sorting equipments (GentleMacs, Tyto, MacsQuant16) are available on the core facility and can be used for your samples preparation → Book or Contact us
RNAseq protocol overview
Source : Shudagar et al., 2017
Single Cell/Single nuclei RNAseq
The core facility is equipped with a Chromium iX (10X Genomics) and provides 3’ mRNA-seq on single cells or nuclei by combining NextGEM technology from 10X Genomics with high-throughput sequencing on NextSeq 2000 (Illumina).
Single-Cell RNAseq (ScRNASeq) and Single-Nuclei RNAseq (SnRNAseq) make it possible to take into account cellular heterogeneity and to characterize rare cell populations by analyzing the expression of genes cell by cell or nucleus by nucleus.
NextGEM technology is based on digital microfluidics allowing manipulation of fluids in microchannels and encapsulation of cells/nuclei with reagents in microdroplets of oil which will behave like microreactors.
The protocol allows preparation of cDNA libraries containing a barcode at the 3' end of the mRNAs contained in each cell/nucleus. The principle is based on the encapsulation of a cell/nucleus in a nanoliter droplet of oil containing a bead, called GEM (Gel Bead-In EMulsions), on which polydT oligonucleotides are attached to capture mRNAs by their polyA tail. The uniqueness is given by the use of a barcode specific to each bead and therefore to each cell. In order to perform single cell analysis, the cells are dispensed by limiting dilution so that 90-99% of the GEMs contain no cell and the rest mainly a single cell.
NextGEM technology overview (10X Genomics)
Source : 10X Genomics
GEM bead schema
Source : 10X Genomics
Each GEM is covered with unique adapter sequences containing a barcode, a UMI and the PolyT sequence. The barcode is the unique bead identifier, and therefore unique for each cell. 10x Genomics offers approximately 750,000 barcodes. The UMI (Unique Molecular Identifiers) is a short random sequence unique to each fragment surrounding the bead. There are therefore several UMI per bead. This identifier is used to avoid amplification bias.
Immediately after the formation of the GEMs, the gel is dissolved, the reagents released and the cell lysed. The reverse-transcription reaction is carried out in the droplets which behave like microreactors. The GEMs are then broken, all the cDNAs pooled and the amplification steps to prepare the libraries are carried out in tubes. The libraries are then purified and sequenced.
Contact us to plan your experiments
Cell sorting equipments (GentleMacs, Tyto, MacsQuant16) are available on the core facility and can be used for your samples preparation ==> Book or Contact usThe core facility perform library preparation, sequencing and primary data analysis with CellRanger.
The preparation of cell/nuclei suspensions is carried out by the project leader.
The quality of the cell/nuclei suspension is the critical step for the ScRNAseq/SnRNAseq success.
The presence of debris and dead cells impacts the encapsulation rate, so the cell viability rate must be greater than 70% → Have a look at the cells or nuclei preparation instructions : Sample-Prep-Cells /Sample-Prep-Nuclei
The number of input cells depends on the number of cells you want to analyze, the average encapsulation rate is 40-60%. Please note that the higher the number of cells/nuclei is, the higher the rate of multiplets per droplet will be (see table below).
It is not recommended to encapsulate more than 5000 cells per well.
10X Genomics recommends an input concentration of 700-1200 cells/µl in a 1X PBS-0.04% BSA.
Source : 10X Genomics
Using Chromium iX it is also possible to work from PFA-fixed cells using the Single Cell Gene Expression Flex protocol, for human or murine samples → See single-cell-gene-expression-flex
Source : 10X Genomics
Ready to Seq Libraries
Contact us to plan shipping and sequencing run of your samples.
Quality controls will be carried out upon arrival of your samples and librairies purification performed if necessary.
Informations needed for sequencing:
- Kit used for library preparation
- Library concentration and size
- Samples names and indexes used
- Sequencing parameters required (SR, PE, length of reads, depth of sequencing, % PhiX…)
Sequencing options on NextSeq 2000
Flow cells can be used for single read (SE) or paired-end (PE) sequencing.
For example, P2 100bp flow cell can be used for SR 100bp or PE 2x50bp sequencing.
Spatial Transcriptomics
