The Human Immune Monitoring Center (HIMC)

Genomics

The HIMC offers genomics services for human and mouse immunology-related projects:  RNA, cfRNA/Exosome, genomic DNA, and cfDNA extraction; Bioanalyzer and qubit analysis services; Human or mouse TCR repertoire analysis (ab and gd TCR, bulk or single cell); human bulk BCR repertoire analysis; microfluidic qPCR arrays; targeted or whole transcriptome RNAseq library preparation (for Illumina platform); and BD Rhapsody single-cell assays, including sample tagging AbSeq, targeted or whole transcriptome gene expression, and/or ab TCR targeting

RNA and DNA extraction and quality control:   RNA/DNA can be extracted from PAXgene RNA or DNA tubes, or from samples preserved in Trizol, or from any biofluids (serum/plasma/CSF, etc.) or FFPE.  Quality/integrity is checked using an Agilent Bioanalyzer chip (small, NANO, or PICO RNA), which automatically determines an RNA Integrity Number (RIN) as well as a visual depiction of electrophoretic results.  The Bioanalyzer can also be used to determine total protein concentration of cell lysates of other protein samples. Qubit and QIAXpert measurements are also part of RNA/DNA QCs metrics.

Single-cell TCRseq: The HIMC offers single-cell TCR repertoire sequencing with targeted mRNAseq (adapted from Han A, et al., Nature Biotechnology 2014).  This 96-well plate-based assay allows the association of cell phenotype (gene expression) with clonality (TCR sequence).  We start from single sorted cells that you provide and return a standard report of gene expression and TCR calls.  We perform all majortypes of single-cell TCR repertoire analysis, including abT and gdT for human and mouse.

Bulk T-cell TCRseq: We offer human and mouse bulk αβT and γδT T-cell TCR repertoire sequencing with modified single cell TCRseq method (Han et al, Nature Biotechnology 2014) and/or 5’RACE αβT TCR profiling methods (commercially available through Takara Bio USA).

Massive single-cell AbSeq+RNAseq: BD Rhapsody is a powerful single cell AbSeq platform for immune monitoring, with the ability to combine protein expression analysis (by AbSeq) with TCR repertoire and/or RNAseq (targeted or whole transcriptome) (adapted from Fan HC, et al., Science 2015). The assay facilitates much deeper insights into immune cell populations (usually a few thousand cells per sample, with Sample Tagging).  All downstream sequencing services will be collaboratively performed at SFGF.

Bulk B-cell BCRseq: We offer human bulk B-cell BCR repertoire sequencing with cutting edge technologies of UMI and PCR-based light and heavy chain CDR3 amplification of all B-cell isotypes (IgG, IgA, IgM, IgE, and IgD) and subtypes of IgG (G1-G4) (adapted from Bashford-Rogers RJM, et al., Nature 2019).

Cell-free RNA and DNA, whole blood gene expression:  For human immunological studies, we provide full service library preparation, concurrently with or without TCR & BCR repertoire, for gene expression at targeted and transcriptome levels. For whole blood sample, including PAXgene blood, we perform hemoglobin RNA and rRNA depletion. For cfRNA and cfDNA samples from serum or plasma, ds cDNA and ds DNA will be generated for library preparation, for high throughput sequencing atSFGF.

Microfluidic qPCR arrays:  The Fluidigm BioMark system allows for the rapid construction of qPCR arrays on a microfluidic chip, using minimal sample volume and minimal pipetting.  The system is compatible with single-cell qPCR.  A set of primers for B- and T-cell specific genes is available in the HIMC, and others can be purchased by customers for use on this platform.

Gene expression and miRNA Microarrays:  We offer a custom "HIMChip" microarray containing ~7000 unique probes for over 3000 human immune-related genes (click here for the list).  Genes were derived from the Immunology Database and Analysis Portal, based on the Entrez Gene, UniProt, KEGG, and various literature sources.  This is a custom Agilent SurePrint HD 8x15K format array, with 8 arrays available per slide.  Whole-genome microarrays, including human and mouse miRNA arrays, and miRNA microarray on the Agilent platform can be done in HIMC collaboratively with the Stanford Functional Genomics Facility (SFGF)    

Technical consultation is also available on experimental design, available tools, data analysis, etc.

 

Some recent publications that used HIMC genomic services:

  1. Tom Erkers, Bryan J Xie, Laura Kenyon, Brian Smith, Mary Rieck, Kent Jensen, Xuhuai Ji,Marina Basina, Samuel Strober, Robert S Negrin, Holden T Maecker, and Everett H Meyer. High-Parametric Evaluation of Human Invariant Natural Killer T Cell to Delineate Heterogeneity in Allo- and Autoimmunity. Blood. January 2020 (in press).
  2.  Saligrama NZhao FSikora MJSerratelli WSFernandes RALouis DMYao WJi XIdoyaga JMahajan VBSteinmetz LMChien YHHauser SLOksenberg JRGarcia KCDavis MMOpposing T cell responses in experimental autoimmune encephalomyelitis. Nature. 2019 Aug;572(7770):481-487. doi: 10.1038/s41586-019-1467-x. Epub 2019 Aug 7.
  3. Guo Luo, Aditya Ambati, Ling Lin, Mélodie Bonvalet, Markku Partinen, Xuhuai Ji, Holden Terry Maecker, and Emmanuel Jean-Marie Mignot. Autoimmunity to hypocretin and molecular mimicry to flu in type 1 narcolepsy. Proc Natl Acad Sci U S A PNAS | vol. 115 | no. 52 | E12323–E12332.
  4. Tang HInoki KBrooks SVOkazawa HLee MWang JKim MKennedy CLMacpherson PCDJi XVan Roekel SFraga DAWang KZhu JWang YSharp ZDMiller RARando TAGoldman DGuan KLShrager JB. mTORC1 underlies age-related muscle fiber damage and loss by inducing oxidative stress and catabolism. Aging Cell. 2019 Jun;18(3):e12943. doi: 10.1111/acel.12943. Epub 2019 Mar 29.
  5. Xuhuai Ji, Igor Goncharov, Miao Zhao, Molly Miranda and Holden T. Maecker. Protein- and Sequencing-based Massively Parallel Single-cell Approaches to Gene Expression Profiling. Bioprotocol. February 5, 2019, e3161.
  6. Winger EE, Reed JL, Ji X, Nicolaides K. Peripheral blood cell microRNA quantification during the first trimester predicts preeclampsia: Proof of concept. PLoS One. 2018 Jan 2;13(1):e0190654. doi: 10.1371/journal.pone.0190654. eCollection 2018.
  7. Winger EE, Reed JL, Ji XEarly first trimester peripheral blood cell microRNA predicts risk of preterm delivery in pregnant women: Proof of concept. PLoS One. 2017 Jul 10;12(7):e0180124. doi: 10.1371/journal.pone.0180124. eCollection 2017.
  8. Glanville JHuang HNau AHatton OWagar LERubelt FJi XHan AKrams SMPettus CHaas NArlehamn CSLSette ABoyd SDScriba TJMartinez OMDavis MM. Identifying specificity groups in the T cell receptor repertoire. Nature. 2017 Jul 6;547(7661):94-98. doi: 10.1038/nature22976. Epub 2017 Jun 21.


User Instructions

1.  Planning your assay:  Please keep in mind the architecture of microarray chips (8 or 12 arrays per slide) when planning experiments and batches.  Please consult with us regarding optimal experimental design (1- or 2-color arrays, replicates, controls).

2.  Collecting your samples:  Good RNA quality and yield are dependent on proper sample handling.  Please see the protocols at the link below, or consult with us before delivering samples.  Avoiding sample contamination is also critical for qPCR experiments.

3.  Running your samples:  An order through our iLab system is required before samples can be run. Turnaround time varies depending on the size of your project and the current workload; smaller projects can usually be completed within 3-4 weeks of order placement and sample delivery.

4.  Data analysis:  You will be notified by email when assays are completed.  Summary reports are provided for Bioanalyzer runs, library prep results, qPCR arrays QCs, and microrray results.  Microarray raw data files are available for download via google drive, or by file transfer in the HIMC (1651 Page Mill Road, Lab 0105).  Additional data analysis services are available for a fee.

Protocols

Please see our Protocols page for detailed lab protocols as well as short descriptions suitable for publications and grant applications.

 

For more information

Contact:

Xuhuai Ji, Genomics Manager

Address:
Human Immune Monitoring Center
1651 Page Mill Road, Lab 0105
Palo Alto, CA 94304

phone:  650-724-1913

email: xuhuai.ji@stanford.edu