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Protein- and Sequencing-based Massively Parallel Single-cell Approaches to Gene Expression Profiling

Xuhuai Ji Xuhuai Ji
IG Igor Goncharov
MZ Miao Zhao
MM Molly Miranda
Holden T. Maecker Holden T. Maecker

Published: Feb 5, 2019 DOI: 10.21769/BioProtoc.3161 Views: 7892

Edited by: Modesto Redrejo-Rodriguez Reviewed by: Lokesh KalekarAnna M. Köhler

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Abstract

Understanding cellular diversity and dynamics in immune cells are critical for elucidating mechanisms of diagnosis, response to therapeutics, and patient prognosis. Our goal is to apply a technically simple approach for gene expression cytometry combining next-generation sequencing with stochastic barcoding of single cells. The BD Rhapsody Single-cell Analysis System was implemented to assess cellular heterogeneity present in 10 Dynabead-sorted human T cell samples. We captured and sequenced over 40,000 single cells across 10 samples. We used oligonucleotide-conjugated antibodies and an immune targeted gene panel that utilizes multiplex PCR for detection of several hundred genes of interest. A combinatorial library of beads bearing cell- and molecular-barcoding capture probes was used to uniquely label transcripts. This allowed for reconstruction of the digital gene expression profile of thousands of individual cells in a single experiment without the need for robotics or automation. We demonstrate the ability of the BD Rhapsody targeted panel to distinguish six different cell types and 18 distinct gene-expression biomarkers, including CLEC4E, CSF2, IL1RN, TPSAB1, CCL19, CCL22, CXCL2, LAMP3, SPP1, and VMO. The BD Rhapsody targeted panel yields more sequencing depth with much fewer (< 2%) sequencing reads per cell. The measurement of specific proteins and transcripts in individual cells is critical for understanding the role of cellular diversity in development, health, and disease.

Keywords: AbSeq search Gene Expression search Targeted RNAseq search Rhapsody search Single Cell RNAseq search Molecular Indexing search

Background

Analyzing individual blood cells can identify their specific role in fighting disease. To understand why cells differ from each other, we need to understand which genes are transcribed (and ultimately expressed as proteins) at a single-cell level. Understanding cellular diversity at both protein and transcriptional levels is critical for elucidating mechanisms of pathogenesis, response to therapeutics, and disease prevention (Han et al., 2014; Landhuis, 2018). The BD Rhapsody Single-cell Analysis System is designed to assess cellular heterogeneity present in biological samples by capturing and sequencing thousands of single cells. The system uses oligonucleotide-conjugated antibodies and a targeted gene panel that utilizes multiplex PCR for detecting hundreds of genes of interest in immunity or cancer (Newell et al., 2012; Wills et al., 2013). A combinatorial library of beads bearing cell- and molecular-barcoding capture probes is used to uniquely label transcripts (UMI).

This allows for reconstruction of the digital gene expression profile of thousands of individual cells in a single experiment without the need for robotics or automation (Shapiro et al., 2013; Fu et al., 2014a and 2014b). Rather than probing the whole transcriptome, this technology focuses on several hundred transcripts, which cuts the number of sequencing reads about tenfold. The BD Rhapsody Targeted Panel can yield more sequencing depth with much fewer (< 2%) sequencing reads per cell and thus facilitates a more affordable single-cell snapshot of heterogeneous subpopulations in single live cells (Fan et al., 2015; Lam et al., 2018; Martin et al., 2018). High sensitivity is demonstrated by the detection of low-abundance transcripts and rare cells. We implemented the Rhapsody Single-cell Analysis System (Reference 1) to assess cellular heterogeneity present in PBMC in this study. BD Rhapsody empowers single-cell data by making experiments easier and more cost-efficient, without using robotics or automation. There are basically no limitations for this approach, as long as there are enough cells (> 1,000 cells) to start with. No other alternative methods for such targeted RNAseq + Abseq are commercially available at the moment.

Materials and Reagents

  1. GilsonTM PIPETMANTM TipackTM filter tips, 100-1,200 μl for BD RhapsodyTM P1200M pipette (Thermo Fisher Scientific, catalog number: F171803G)
  2. GilsonTM PIPETMANTM TipackTM Racked pipet tips, 500-5,000 μl for BD RhapsodyTM P5000M pipette (Thermo Fisher Scientific, catalog number: F161370G)
  3. Falcon® tube with cell strainer cap (Thermo Fisher Scientific, catalog number: 352235)
  4. DNA LoBind tubes, 1.5 ml (Eppendorf, catalog number: 0030108051)
  5. DNA LoBind tubes, 5 ml (Eppendorf, catalog number: 0030108310)
  6. Low retention filtered pipette tips, 10 μl (Gilson PIPETMAN, catalog number: 1222Q28; RAININ, catalog number: 17015169)
  7. Low retention filtered pipette tips, 200 μl (Gilson PIPETMAN, catalog number: 1222Q31; RAININ, catalog number:17014963)
  8. Low retention filtered pipette tips, 1,000 μl (Gilson PIPETMAN, catalog number: 1222Q34; RAININ, catalog number: 17015122)
  9. QubitTM assay tubes (Thermo Fisher Scientific, catalog number: Q32856)
  10. 0.2 ml PCR 12-strip tubes (Axygen, catalog number: 14-222-259)
  11. 10 ml sterile serological pipettes (Fisher Scientific, catalog number: 13-676-10F)
  12. 15 ml tubes (Conical Centrifuge Tubes, Fisher Scientific, catalog number: 14-959-53A)
  13. Waste collection container (BD Biosciences, PN 650000090)
  14. Agencourt® AMPure® XP magnetic beads (Beckman Coulter, catalog number: A63880)
  15. BDTM Stain Buffer (FBS) (BD Biosciences, catalog number: 554656)
  16. Calcein AM cell permeant dye (Thermo Fisher Scientific, catalog number: C1430)
  17. DRAQ7TM, 0.3 mM (BD Biosciences, catalog number: 564904)
  18. Dimethyl sulfoxide (DMSO) (Corning, catalog number: 25950CQC)
  19. 70% ethyl alcohol or 70% isopropyl alcohol
  20. Ethyl alcohol, Pure (200 proof, molecular biology grade) (Sigma-Aldrich, catalog number: E7023-500ML)
  21. 1x RBC Lysis Buffer (Thermo Fisher Scientific, catalog number: 00-4333-57)
  22. Phosphate-buffered saline (calcium- and magnesium-free) (Gibco, catalog number: 10-010-023)
  23. Premoistened cleaning wipes with 70% ethyl alcohol or 70% isopropyl alcohol
  24. BD Rhapsody Cartridge Reagent Kit (BD Genomics, PN 633731)
  25. BD Rhapsody Cartridge Kit (BD Genomics, PN 633733)
  26. BD Rhapsody cDNA Kit (BD Genomics, PN 633732)
  27. BD Rhapsody Targeted Amplification Kit (BD Genomics, PN 633734)
  28. BD Single-cell Multiplexing Kit–Human Sample Tag (12) Component (BD Genomics)
  29. BD Single-cell Multiplexing Kit Library Amplification Component (BD Genomics, PN 633782)
  30. BD RhapsodyTM Immune Response Panel Hs (BD Genomics, PN 633750)
  31. QubitTM dsDNA HS Assay Kit (Thermo Fisher Scientific, catalog number: Q32851)
  32. Agilent Bioanalyzer DNA High Sensitivity Kit (Agilent Technologies, catalog number: 5067-4626)
  33. 10% bleach (Thermo Fisher Scientific, catalog number: 50-163-3379)
  34. cDNA master mix prep (see Recipes)
  35. Exonuclease I master mix (see Recipes)
  36. PCR1 reaction master mix (see Recipes)
  37. PCR Program 1 (see Recipes)
  38. Targeted PCR2 reaction master mix (see Recipes)
  39. Sample Tag PCR2 reaction master mix (see Recipes)
  40. Targeted final amplification master mix (see Recipes)
  41. Sample Tag final amplification master mix (see Recipes)
  42. PCR Program 2 (see Recipes)

Equipment

  1. Pipettes (P2, P10, P20, P200, P1000) (RAININ Single channel Pipet-Lite XLS, P2: catalog number:17014393; P10: catalog number: 17014388; P20: catalog number: 17014392; P200: catalog number: 17014391; P1000: catalog number: 17014382)
  2. Multi-channel pipette, P2-20 μl or P20-200 μl (RAININ Multichannel Pipet-Lite XLS, P2-20: catalog number: 17013808; P20-200: catalog number: 17013810)
  3. Pipet-Aid (Drummond, catalog number: 13-681-06)
  4. Illumina HiSeq 4000 sequencer (Illumina, HiSeq 4000 system)
  5. Laminar flow hood (AirClean System AC600, catalog number: 36-100-4377)
  6. Digital timer (Fisherbrand, catalog number: 06-662-5)
  7. Vortexer (Fisherbrand, catalog number: 02-215-414)
  8. 6-Tube Magnetic Separation Rack for 1.5 ml tubes (New England Biolabs, catalog number: S1506S)
  9. Large magnetic separation stand (V&P Scientific, catalog number: VP 772FB-1)
  10. Clear acrylic cylinder adapter for 15 ml tube magnet (V&P Scientific, catalog number: VP 772FB-1A)
  11. Low-profile magnetic separation stand for 0.2 ml, 8-strip tubes (V&P Scientific, or Clontech, catalog numbers: VP 772F4-1 or 635011)
  12. Improved Neubauer Hemocytometer (INCYTO, model: DHC-N01-5)
  13. BD Rhapsody Scanner (BD Genomics, PN 633701)
  14. Sample Loading Station (BD Genomics, PN 63370)
  15. ThermoMixer® C (Eppendorf, catalog number: 5382000023)
  16. SmartBlockTM Thermoblock 1.5 ml (Eppendorf, catalog number: 5360000038)
  17. QubitTM 3.0 Fluorometer (Thermo Fisher Scientific, catalog number: Q33216)
  18. VWR® Advanced Mini Dry Block Heater with Heated Lid (VWR, catalog number: 10153-348)
  19. 2100 Bioanalyzer (Agilent Technologies, catalog number: G2940CA)
  20. Thermal cycler with heated lid (Thermo Fisher Scientific SimplAmp, catalog number: A24811)
  21. Water bath or incubator at 37 °C (Thermo Scientific, catalog number: TSCOL19)
  22. Microcentrifuge for 1.5-2.0 ml tubes (Thermo Scientific, catalog number: 75004061)
  23. Microcentrifuge for 0.2 ml tubes (Southwest Science, catalog number: SC1006-MBlue)
  24. Centrifuge and rotor for 15 ml tubes (Eppendorf 5417C, catalog number: EP-5417C; Rotor, catalog number: A-4-44)
  25. Barcode reader with keyboard wedge, USB 2.0 or 3.0 compatible (Brady Code Reader 3600, catalog number: 19-104-473)

Software

  1. BD RhapsodyTM Analysis pipeline (on Seven Bridges Genomics platform)
  2. BDTM Data View (run under MATLAB)
  3. MATLAB Runtime (R2017b, 9.3, for Windows) (https://www.mathworks.com/products/compiler/matlab-runtime.html)

Procedure

Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.

Category

Immunology > Immune cell function > General

Molecular Biology > RNA > Transcription

Cell Biology > Cell-based analysis > Gene expression

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