FAQ’s

The Genomics Shared Resource (GSR) is located in Rubin 670 in the Norris Cotton Cancer Center, Lebanon, NH. We strongly encourage all users to schedule a free consultation with a GSR scientist before beginning a new project. Through this consultation, we can help you select the most appropriate technology and workflow for your research questions and provide pricing information to ensure the service fits within your budget. You can reach us at GMBSR@groups.dartmouth.edu or by phone at (603) 646-5502.

All orders are placed through the RaDar system. Please complete any required order forms for the service and provide an active account number. It is also recommended to email a member of the Shared Resource to confirm receipt of the order. Pricing information and a cost estimation tool are available on the Price Estimation Tool page. New external clients should contact Chris Lytle or Fred Kolling for RaDar registration information.

The GSR has created an online Price Estimation Tool to help estimate costs for common assays. Prices listed on the service pages reflect internal Dartmouth rates for FY2026 and are based on standard batch sizes; actual prices may vary. External users and those with large projects should contact the core directly for customized pricing. We strongly encourage all users to schedule a free consultation with a GSR scientist to review experimental design and obtain an accurate quote before starting a project.

We strongly recommend a minimum of 4 biological replicates per experimental condition for all experiments. Adequate replication is essential for statistical power and reproducibility, particularly in differential expression analyses. The optimal number of replicates may vary depending on the expected effect size, variability of the biological system, and the specific analysis being performed. Please consult with a GSR scientist to determine the appropriate experimental design for your study.

Yes. The GSR performs DNA and/or RNA extraction from cell pellets, tissue chunks, and FFPE samples. For cells and tissues, samples should be snap-frozen on dry ice or homogenized in Qiagen RLT buffer and stored at −80°C prior to submission. Cell input range is 10,000–10,000,000 cells; tissue input range is 1–10 mg. FFPE extraction is also available, though simultaneous DNA + RNA extraction from FFPE typically yields lower amounts than extracting either nucleic acid alone. Please contact the GSR before submitting samples to coordinate timely processing.

The GSR uses three complementary QC instruments: (1) Invitrogen Qubit Flex — fluorescence-based quantification of DNA or RNA with high sensitivity, especially for low-concentration samples (minimum 3 µL required). (2) Agilent Fragment Analyzer — high-resolution, 96-well plate format sizing and quality assessment of DNA (20 bp–50 kb) and RNA (15 nt–6,000 nt), including gDNA, NGS libraries, smRNA, and total RNA, down to 200 pg/µL sensitivity. (3) Agilent TapeStation — rapid quality assessment of DNA and RNA including gDNA, NGS libraries, smRNA, and total RNA. For FFPE RNA samples, a DV200 score is calculated from the Fragment Analyzer to identify potentially problematic samples before library preparation.

Yes. FFPE samples can be used for NGS and microarray workflows, though they often have degraded nucleic acids that can affect performance. The GSR applies rigorous QC to FFPE samples before proceeding: RNA is evaluated with the Fragment Analyzer to calculate a DV200 score (the percentage of RNA fragments >200 nt), and FFPE DNA is assessed using a qPCR-based quality assay benchmarked against a validated control. For methylation arrays, FFPE DNA can undergo an additional restoration step. Single cell RNA-seq of fixed or FFPE samples is supported via the 10x Genomics Flex (Fixed RNA Profiling) assay, with fixed samples storable at −80°C for up to 6 months.

All services provided by the GSR are supported by an NCI Cancer Center Support Grant (5P30CA023108), which must be cited in any publications or other materials using data generated in the core. Single cell sequencing services are additionally subsidized by a COBRE grant (P20GM130454), which must be cited in all publications and materials using single cell data generated in the core. Detailed acknowledgement language and facility descriptions for grant applications are available on the Acknowledgements and Authorship page.

Data generated in the GSR is stored on the Dartmouth DartFS file storage system, which can be accessed through the Discover/Andes/Polaris computing environments or mounted as a network drive on your computer. Each lab is provided a dedicated folder with access restricted to NetIDs approved by the PI. If a lab does not use DartFS, or if data needs to be shared with collaborators outside Dartmouth, a web server with download links is available upon request. Raw data is stored as demultiplexed FASTQ files in a read-only format for 5 years. An email alert will be sent prior to deletion; users are encouraged to copy data to their own storage promptly after delivery.

Yes. The GSR accepts samples from external academic, government, and commercial users. External users are subject to different pricing than internal Dartmouth users; please contact the GSR directly for an external rate quote. New external clients should contact Chris Lytle or Fred Kolling for information on RaDar registration and account setup. Data from external users is delivered via a web server with download links if DartFS access is not available. All external projects are subject to the same consultation, QC, and grant acknowledgement requirements as internal projects.

The GSR offers four bulk RNA-seq workflows: (1) Ribodepletion RNA-seq — provides full-length coverage of mRNAs and long non-coding RNAs; the only option for prokaryotic samples; recommended depth ~30M reads/sample. (2) 3′ End-Counting RNA-seq — a cost-effective option for differential expression from eukaryotic samples with well-annotated genomes (human, mouse, rat); cannot detect splice isoforms; ~10M reads/sample recommended. (3) miRNA/smRNA-seq — captures small RNAs via direct adapter ligation with UMIs to minimize PCR bias; 1–10M reads/sample. (4) Low Input RNA-seq — designed for samples with 250 pg–10 ng RNA input, using probe-based rRNA removal and UMIs. Consult with the GSR or use the online estimation tool to choose the best workflow.

RNA can be isolated from 1,000 to >1 million cells and from most tissue and fluid types. FFPE samples can be processed but reduced yield and quality are expected. Small numbers of cells (<100,000) should be FACS sorted or deposited into 200 µL Qiagen RLT buffer and stored at −80°C until submission. Larger cell numbers can be pelleted and resuspended in 200 µL RLT buffer and stored at −80°C. Tissue samples can be stored in RNAlater at −80°C until submission. Please coordinate with the GSR to ensure timely processing and prevent RNA degradation.

Input requirements vary by workflow. Ribodepletion RNA-seq typically requires 100 ng–1 µg of total RNA (RIN >7 recommended, though degraded samples may still yield usable libraries). 3′ End-Counting RNA-seq works best with high-integrity RNA (RIN >8) and a standard input of ~100 ng total RNA. miRNA/smRNA-seq uses 100 ng–1 µg total RNA. Low Input RNA-seq is designed for samples with only 250 pg–10 ng total RNA. All RNA samples are QC’d with the Qubit Flex and Fragment Analyzer or TapeStation before library preparation. Samples that do not meet minimum quality thresholds will be flagged and discussed with the user before proceeding.

The GSR offers two primary DNA-seq services: (1) Standard DNA-seq — prepares sequencing libraries from intact genomic DNA (whole genome sequencing) or sheared DNA (e.g., ChIP-seq, CUT&RUN). Intact gDNA is fragmented using a Covaris E220 Evolution sonicator before adapter ligation and amplification. Up to 192 samples can be uniquely barcoded. (2) Target Capture / Exome Sequencing — uses IDT xGen reagents for whole exome or custom targeted sequencing panels, preferred over PCR-based approaches for large targeted studies. IDT’s Exome v2 panel is stocked by the core. A sequencing coverage calculator is available to help determine requirements.

For standard DNA-seq (whole genome or sheared DNA for ChIP-seq/CUT&RUN), a minimum of 1–10 ng of DNA is typically sufficient, with higher input (>100 ng) preferred for optimal library complexity. DNA is assessed by Qubit and Fragment Analyzer prior to library preparation. For Target Capture / Exome Sequencing, at least 100–200 ng of high-molecular-weight genomic DNA is recommended; the IDT xGen capture workflow requires adequate input to ensure on-target enrichment. FFPE DNA samples are evaluated by a qPCR-based quality assay before proceeding. Please consult with the GSR if you are unsure whether your samples meet the minimum input requirements.

Yes. The GSR can prepare sequencing libraries from sheared chromatin IP (ChIP-seq) or CUT&RUN samples that users provide. For these workflows, users perform the immunoprecipitation or CUT&RUN reaction themselves and submit the resulting DNA to the GSR for end-repair, adapter ligation, and amplification using the standard DNA-seq library preparation workflow. Samples should be provided in a volume of at least 10–20 µL of eluted DNA. The GSR will perform Qubit quantification and Fragment Analyzer sizing QC before proceeding. Please contact the GSR to discuss input requirements and experimental design before beginning your experiment.

Recommended sequencing depths vary by application: Ribodepletion RNA-seq: ~30M reads/sample for mammalian genomes (less for small prokaryotic genomes). 3′ End-Counting RNA-seq: ~10M reads/sample. miRNA/smRNA-seq: 1–10M reads/sample. Low Input RNA-seq: ~30M reads/sample. For whole genome and exome sequencing, depth requirements depend on the application and genome size; use the GSR’s coverage calculator or consult with the core. The GSR operates an Illumina NextSeq 2000 (100M–1.2B reads/run) and an Illumina MiniSeq (7M–30M reads/run) to accommodate a wide range of project scales.

The GSR offers Illumina array-based DNA methylation profiling: (1) Illumina Methylation EPIC — profiles over 850,000 CpG and non-CpG sites across the human genome for quantitative differential methylation analysis (8 samples per array). (2) Illumina Mouse Methylation — profiles over 285,000 CpG sites in the mouse genome (12 samples per array). (3) FFPE Restoration for EPIC Arrays — a qPCR-based quality scoring and restoration workflow for FFPE DNA samples prior to array processing. (4) Customer-Ordered Illumina Infinium Arrays — the GSR will process any Illumina Infinium array ordered by the user. All services include DNA quantification, bisulfite conversion, array labeling, scanning, and data delivery.

Yes. You can provide pre-pooled and normalized libraries that are ready for sequencing, or the GSR can perform library QC, normalization, and pooling on your behalf. For pre-pooled libraries, the GSR is not responsible for issues with sample balancing or data yield. You should also provide an Illumina sample sheet or a list of the index sequences used for each sample so the GSR can deliver demultiplexed FASTQ files. Please contact the GSR before submitting self-prepared libraries to confirm compatibility and sequencing requirements.

Yes. The GSR is happy to work with you and the vendor to design custom sequencing or microarray panels suited to your needs. For custom target capture panels, users should use the IDT Design Tool to create and order panels; the GSR will perform all downstream processing. For custom Illumina Infinium arrays, users order the arrays from Illumina and the GSR performs DNA quantification, labeling, scanning, and data delivery. The user is responsible for ordering custom reagents while the GSR charges for common reagents and labor. Please consult with GSR staff early in the design process.

The GSR offers a comprehensive suite of single cell services using the 10x Genomics Chromium X platform, including: 3′/5′ scRNA-seq, ATAC-seq (open chromatin profiling), Multiome (simultaneous RNA+ATAC from the same nucleus), CITE-seq (surface protein + RNA), TCR/BCR V(D)J sequencing, Gene Expression Flex (fixed/FFPE samples), and sample multiplexing. The GSR also offers the Honeycomb HIVE picowell-based platform as an alternative scRNA-seq option. All single cell projects require a free pre-project consultation. Services include sample QC, all library preparation, and downstream QC steps. Each 10x lane can capture 100–20,000 cells.

For most 10x Genomics workflows, users must provide a high-quality, viable single cell or single nucleus suspension. Cell viability should ideally be >85%, and cell concentrations should be verified with the Nexcelom Cellometer K2 fluorescent cell counter. The GSR offers tissue dissociation services to generate single cell suspensions from fresh/frozen tissues, as well as nuclei isolation for ATAC-seq and Multiome workflows. For fixed samples using the 10x Flex protocol, the GSR can perform sample fixation with storage at −80°C for up to 6 months. All single cell projects require a prior consultation with GSR staff.

The GSR offers tissue processing services to help users generate high-quality single cell or single nucleus suspensions for 10x Genomics workflows. Services include: (1) Tissue dissociation into a single cell suspension — off-the-shelf dissociation protocols are available for a number of commonly used tissue types; tissues requiring protocol optimization may incur additional charges. (2) Nuclei isolation from fresh or frozen tissue — required for ATAC-seq and Multiome workflows. These services are performed using the Cytiva VIA Extractor, an automated system providing full control over temperature, homogenization intensity, and run time for gentle and consistent tissue processing.

Yes. The GSR offers sample multiplexing for 10x Genomics workflows using On-Chip Multiplexing (OCM) or hash-tag oligonucleotide (HTO) approaches such as MULTI-seq or Total-seq. The 10x 3′/5′ RNA-seq OCM service allows up to 4 samples to be combined and processed in a single lane (up to 20,000 cells total), significantly reducing per-sample costs. The 10x Gene Expression Flex assay also supports multiplexing of 1, 4, or 16 barcoded samples per lane, with increasing economies of scale. Please consult with the GSR to determine which multiplexing strategy is best suited to your experimental design.

The Honeycomb HIVE is a picowell-based single cell RNA-seq platform that offers a flexible alternative to the 10x Genomics droplet approach. Each HIVE can capture 500–60,000 cells (30,000 recommended). A key distinguishing feature is that the user loads the HIVE themselves — cells are loaded directly onto the device, which can then be sealed and stored for months before being submitted to the GSR for library preparation and sequencing. This is particularly useful for time-sensitive samples or when batching experiments over time. Recommended sequencing depth is 25,000–50,000 reads per cell.

Recommended sequencing depths vary by assay: 10x scRNA-seq (3′/5′): ~30,000 reads/cell. 10x ATAC-seq: ~25,000 reads/cell. 10x Multiome: ~25,000 reads/cell each for RNA and ATAC (50,000 total/cell). 10x Flex: ~20,000 reads/cell. Honeycomb HIVE: 25,000–50,000 reads/cell. For Library Add-Ons (TCR/BCR, CITE-seq, etc.), an additional ~5,000 reads/cell per library type is typically required. Actual depth requirements may vary depending on cell type, RNA content, and research goals. The GSR will recommend appropriate sequencing depth during the project consultation.

Yes. The GSR offers free instrument training on the 10x Genomics Chromium X for users who need access outside of standard core hours (Monday–Friday, 8am–5pm). Training takes approximately 2 hours and covers the principles of the 10x microfluidic workflow as well as hands-on practice using non-biological “dummy” reagents. Upon successful completion, users are granted access to the GSR lab and the instrument reservation calendar through RaDar. Users who operate the instrument independently are still required to follow all 10x Genomics protocols and consult with GSR staff for guidance.

A range of additional library types can be added to standard 10x 3′/5′ RNA-seq experiments via a Library Add-On service. These include: TCR/BCR V(D)J sequencing (requires 5′ RNA-seq), CITE-seq (surface protein profiling using antibody-derived tags), sample multiplexing via MULTI-seq or Total-seq hashtags, and other custom assays. Each additional modality requires a Library Add-On per sample and additional sequencing (typically ~5,000 reads/cell per library type). The Curiox Laminar Wash system is used for gentle, centrifuge-free cell washing in CITE-seq and multiplexing workflows to maximize cell retention and data quality.

Single cell genomics projects typically take 2–4 weeks from sample acceptance to data delivery, depending on complexity, batch size, and current demand. Projects requiring tissue dissociation, nuclei isolation, or sample fixation may require additional lead time. The GSR will provide a more specific timeline during your initial consultation and will keep you informed of progress throughout the project. To avoid delays, please coordinate with the GSR well in advance — especially for time-sensitive samples such as primary tissues that must be processed immediately after collection.

Successful single cell experiments depend heavily on sample quality and experimental planning. Key considerations include: ensuring cell viability >85% at the time of loading; targeting an appropriate cell concentration (verified with the Cellometer K2); selecting the correct 10x workflow for your cell/tissue type and research question; planning adequate biological replication; and coordinating with the GSR to schedule instrument time. The GSR recommends scheduling a free pre-project consultation early in the planning process. For ATAC-seq and Multiome workflows, specific nuclei isolation procedures must be followed and should be discussed with GSR staff in advance.

The GSR offers four spatial transcriptomics approaches: (1) Visium Fresh Frozen — PolyA-based capture for fresh/frozen tissue sections; applicable to human, mouse, rat, zebrafish, and more. (2) Visium FFPE — probe-based whole transcriptome profiling of FFPE sections using the CytAssist; ~5,000 or 14,000 barcoded 55 µm spots per capture area. (3) Visium HD — highest resolution Visium option, with 2×2 µm barcoded squares providing near single cell–scale spatial resolution on FFPE sections. (4) Xenium — in situ analysis platform for imaging hundreds to thousands of targeted genes at subcellular resolution using probe hybridization/ligation. Consult the GSR to determine the best approach.

It is applicable to a wide range of species. Visium FFPE uses a probe hybridization approach (whole transcriptome human or mouse probes) on standard FFPE sections, processed via the 10x Genomics CytAssist instrument. FFPE sections undergo clinical-grade H&E staining and 40× whole-slide imaging before probe transfer. Each capture area contains either 5,000 (6.5×6.5 mm) or 14,000 (11×11 mm) barcoded 55 µm spots.

The 10x Genomics Xenium is an in situ spatial analysis platform that differs fundamentally from Visium. Rather than capturing and sequencing RNA from tissue sections, Xenium uses probe hybridization and ligation directly within the tissue to detect hundreds to thousands of targeted genes at subcellular resolution. This enables precise spatial mapping of transcripts while preserving tissue morphology. Custom or off-the-shelf probe sets are available from 10x Genomics. The Xenium workflow includes sample preparation (probe hybridization and ligation) as a separate billable step before instrument analysis. Xenium is ideal when subcellular resolution and large gene panel imaging are required.

All spatial genomics assays at the GSR are performed in collaboration with the Pathology Shared Resource (PSR). For Visium FFPE and Visium HD, the PSR cuts 5 µm FFPE sections onto standard histology slides, performs clinical-grade H&E staining, and provides 40× whole-slide imaging. For Visium Fresh Frozen, the PSR cuts 10 µm cryosections directly onto the Visium capture slides. For Xenium, the PSR cuts sections onto Xenium slides. H&E staining and imaging are performed in the Single Cell Genomics Core. Users should plan for PSR coordination when designing their spatial genomics projects, as tissue sectioning scheduling may affect overall turnaround time.

Spatial genomics projects typically take 3–6 weeks from sample acceptance to data delivery, depending on the assay, tissue type, and current demand. The PSR tissue sectioning schedule and instrument availability may also affect timing. The GSR will provide a more specific timeline during the initial consultation. We strongly recommend contacting the GSR well in advance of your desired start date, particularly for Visium Fresh Frozen projects that require optimization.