Guidelines for droplet-based single-nuclei Multiome (RNA + ATAC; 10X Genomics)
GENERAL GUIDELINES FOR SAMPLE PREPARATION
The single-nuclei Multiome assay allows to simultaneously profile the transcriptome and regulatory landscape of the same nucleus.
CHECKLIST CRITICAL POINTS
A successful droplet-based single-nuclei sequencing experiment in the 10x Chromium system is highly dependent on the quality of the nuclei. Therefore, it is important to be aware of some critical aspects of sample prep to ensure a proper and good preparation of the sample:
1. Nuclei should be intact, thus a proper nuclei isolation is crucial
Optimization of the nuclei isolation protocol is important to obtain intact nuclei. In general, we recommend to be as gentle as possible to avoid overlysing. Buffer composition, lysis conditions, types of mechanical dissociation (if needed), and time/speed of spinning of the dissociated nuclei solution are some of the most relevant variables that can affect nuclei isolation. One should carefully test these conditions prior to running the experiment, and keep in mind that different tissue/cell types need to be optimized independently.
A good quality control to assess the isolation procedure is a visual inspection of the nuclei under a microscope (minimum 40x objective), after staining an aliquot with DNA-intercalating dyes such as propidium iodide, or vital dyes such as trypan blue. Nuclei should have a roundish shape, the nuclear membrane should be intact, nuclei shouldn’t be clumped together, and DNA should not be leaking out. Ideally, the solution should be fairly clear of cell debris. To obtain a more pure nuclei solution, it is possible to perform a low-speed density gradient centrifugation (e.g. Percoll, sucrose, or iodixanol gradients). Alternatively,10X offers a commercial solution for nuclei isolation.
* When nuclei are overlysed, the membrane is compromised and the chromatin likely will no longer be under its native state; in this case, “natively closed” genomic loci can be tagmented, leading to increased noise and decreased specificity of the data.
2. Nuclei suspension should be clean of debris and aggregates
Avoid long sorting times (longer than 1h) as this may lead to loss of stability. Maximum sorting time depends on cell type (sensitivity or robustness of nuclei to experimental procedures and conditions).
It is crucial that the nuclei suspension is clean of debris, and that nuclei are not clumped together. These are frequent causes of clogging of the 10x chip, which lead to loss of the sample.
Use of RNase inhibitor is mandatory for the Multiome assay. You should add 1 U/µl RNase Inhibitor to all buffers; please see specific recommendations from 10x Genomics.
3. Observations
Approximately 65% of the nuclei loaded into the 10x microfluidic chip are encapsulated.
As in every 10x assays, there is a small expected portion of multiplets, i.e., more than one nucleus encapsulated in a single bead. The estimated rate of multiplets is 0.8% for every 1.000 nuclei loaded.
INPUT MATERIAL
Type & media
Nuclei suspension, as clean as possible from debris and no aggregates or clumps. If no sorting is done, we recommend filtering the nuclei suspension with a 30-40 µm strainer; alternatively doing a density gradient centrifugation (e.g. Percoll, sucrose, Iodixanol, etc).
One suggested protocol for nuclei isolation can be found at the end of these guidelines. In addition, we recommend to go through the demonstrated protocols for nuclei isolation from 10X Genomics. .
Nuclei should be delivered in Diluted Nuclei Buffer (DNB) from 10x Genomics. We provide aliquotes of 20x stock Nuclei Buffer, which should be diluted to 1x with nuclease-free water with 1mM DTT and 1 U/µl RNase inhibitor.
Amount & volume
The maximum number of nuclei that can be sequenced is 10.000. When aiming for this number, please deliver nuclei in a concentration of 4.000-8.000 nuclei/µl . Please speak to us beforehand, if it is not possible to reach this concentration.
Before loading, we inspect nuclei in the microscope to check for quality and estimate concentration, thus we ask you to bring a few additional µl for this.
When aiming for lower number of nuclei, please adjust the nuclei concentration following the 10x reference table at the end of this document.
* We recommend using LoBind or BSA-coated tubes (1% BSA [RNase-free]-PBS) for sample prep, to reduce nuclei loss (avoids nuclei sticking to tube wall). We can provide BSA-coated tubes for you, or you can prepare them yourself following our recipe. If using BSA-coated tubes, make sure all BSA is removed before adding the sample to the tube.
* Use of RNase inhibitor is critical! Please add 1 U/µl RNase Inhibitor to all buffers.
NOTES
We perform a visual inspection of the nuclei under the microscope, to check if they are viable, intact, and to estimate concentration. This step is only done if there are enough nuclei in the sample.
Nuclei labelling for sample multiplexing is currently not supported by 10X Genomics.
SEQUENCING
Paired-end sequencing, 2x100bp reads (different read lengths can be requested). Sequencing depth generally depends on cell type, but a good starting point is 30k reads/nuclei for transcriptome part and 25k reads/nuclei for the ATAC part.
Technical/biological replicates are dependent on experimental design and research question. Specificities of the species, sample, or project design, can influence the optimal sequencing coverage. We are glad to provide guidance with this.
BIOINFORMATICS ANALYSIS
The Multiome analyses pipeline gives information about the number of nuclei that were sequenced, peak detection, count matrix for peaks and transcripts, clustering analysis, and differential accessibility/gene expression between clusters. The pipeline is based on the cellranger workflow from 10x Genomics; more information can be found here.
In case you need further support, or any other type of information, we can arrange a meeting with one of our bioinformatics experts to discuss the experiment.
SAMPLE SUBMISSION
Because nuclei have to be loaded fresh into the microfluidic system, we need to process the samples immediately upon receiving. Therefore, we ask users to contact us 2 weeks in advance (or earlier, if you are planning a large experiment) so we can schedule a date and time for the immediate processing of the samples.
Please bring samples in the scheduled time, since our planning of downstream processes is done accordingly. Usually, samples should be delivered by 13h latest, preferrably between Tuesday-Thursday. If your biological system/experimental setup does not allow to schedule the delivery earlier, please speak to us beforehand to agree on a date/time that suits both sides.
We strongly recommend using LoBind tubes or BSA-coated tubes for low nuclei numbers (<100k nuclei in total). Use of non LoBind tubes will drastically reduce yields.
Providing project and sample information via the Rosalind Client Portal
Please log in to Rosalind to enter all necessary project information making use of the “Project Description” field during the project set-up dialogue. Give all relevant details about project design, sample preparation method and how the samples are eluted and/or preserved. Sample-specific information such as concentration, volume, used preparation protocols etc. should be provided when the samples are added to the created project.
Please also indicate if you require a bioinformatics analysis during project setup.
To submit samples, you must be registered in our Rosalind client portal. Therefore, please send an e-mail to genomecenter@tu-dresden.de with your contact details (name, working group, working group leader, your e-mail address and telephone number). This will enable us to set up an account for you. After you have received an automatic e-mail, please log in to Rosalind with your login details. If needed, we can reset your login details.
Following up project progress
You can accompany the status of your project in our Rosalind Client Portal. Documents with information about the preparation method and quality control of samples and libraries are provided for your project.
ADDITIONAL RESOURCES
10x Genomics has an incredibly helpful Q&A section, we highly recommend searching and/or browsing to get more details about sample preparation procedures, general questions about available assays, and details about data analysis.