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BRAIN Publication-derived

Rubella virus tropism and single-cell responses in human primary tissue and microglia-containing organoids

Source Popova et al., 2023 · University of California, San Francisco · 10.7554/elife.87696

👤 Galina Popova, Hanna Retallack, Chang N Kim, Albert Wang, David Shin, Joseph L DeRisi, Tomasz Nowakowski ⏱ 45 days 📋 18 phases 🧫 Human iPSC

Abstract

This protocol establishes methods for generating brain organoids with engrafted primary human microglia and infecting them with rubella virus to study viral tropism and neuroimmune responses. The system reveals that microglia are the predominant target of RV infection and that microbial presence modulates interferon responses in neural progenitor cells and neurons, providing insights into congenital rubella syndrome pathogenesis.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

143 steps across 18 phases

Viral stock generation Prior to infection experiments
  1. 1 Linearize RV-M33 plasmid
  2. 2 Purify RNA from transcription reaction
  3. 3 Polyadenylate RNA
  4. 4 Cap RNA
  5. 5 Transfect Vero cells
  6. 6 Harvest culture media at 72 hr post-transfection
  7. 7 Passage virus onto fresh Vero cells
  8. 8 Harvest and clarify viral stock
  9. 9 Titer viral stock
RV-GFP reporter virus generation Prior to infection experiments
  1. 1 Design GFP-expressing RV construct
  2. 2 Prepare DNA oligos for CRISPR system
  3. 3 Cut plasmid with nCas9 and guides
  4. 4 PCR amplify sfGFP sequence
  5. 5 Insert sfGFP into cut plasmid
  6. 6 Generate RV-GFP viral stocks
  7. 7 Validate GFP expression
Primary prenatal brain tissue preparation Day 0-1
  1. 1 Obtain and prepare cortical brain tissue
  2. 2 Remove blood vessels and meninges
  3. 3 Embed tissue in low-melting-point agarose
  4. 4 Section tissue perpendicular to ventricle
  5. 5 Transfer slices to culture inserts
  6. 6 Culture brain slices
Brain slice infection with rubella virus Day 2-5
  1. 1 Prepare RV inoculum
  2. 2 Apply RV to brain slices
  3. 3 Replace with fresh culture media
  4. 4 Incubate infected slices
  5. 5 Prepare controls
Primary human microglia purification Day 0-1
  1. 1 Prepare brain tissue in aCSF
  2. 2 Mince tissue
  3. 3 Enzymatic digestion
  4. 4 Mechanical trituration
  5. 5 Filter cell suspension
  6. 6 Pellet cells
  7. 7 Wash cells
  8. 8 Resuspend in MACS buffer and add DNAase
  9. 9 Incubate with CD11b antibody
  10. 10 Wash cells in MACS buffer
  11. 11 Load cells onto MACS column
  12. 12 Wash column
  13. 13 Elute microglia
  14. 14 Pellet and count microglia
2D microglia culture and co-culture preparation Day 0-5
  1. 1 Prepare culture plates
  2. 2 Add extracellular matrix proteins
  3. 3 Plate MACS-purified microglia
  4. 4 Culture microglia monocultures
  5. 5 Prepare non-microglial cell fractions for co-culture
  6. 6 Set up co-culture experiments
  7. 7 Culture co-culture conditions
Transwell co-culture system setup Day 0-5
  1. 1 Prepare transwell plates
  2. 2 Plate microglia in bottom chamber
  3. 3 Culture microglia in bottom chamber
  4. 4 Prepare non-microglial cells in transwell
  5. 5 Culture transwell co-culture
2D microglia culture infection with RV Day 2-5
  1. 1 Prepare RV inoculum for 2D cultures
  2. 2 Apply RV inoculum to cultures
  3. 3 Replace inoculum with fresh media
  4. 4 Incubate infected cultures
  5. 5 Prepare controls
iPSC culture and cerebral organoid generation Days 0-35
  1. 1 Maintain iPSC cultures
  2. 2 Dissociate iPSCs for organoid formation
  3. 3 Transfer to ultra-low attachment plates
  4. 4 Begin neural induction (Days 0-9)
  5. 5 Transfer to shaker (Day 9)
  6. 6 Expansion phase (Days 9-25)
  7. 7 Differentiation phase (Days 25-35)
  8. 8 Maturation phase (Day 35 onward)
Microglia transplantation into organoids Day 35-42
  1. 1 Prepare MACS-purified microglia
  2. 2 Add microglia to organoids
  3. 3 Allow microglia engraftment
  4. 4 Return to shaker and standard maintenance
Organoid infection with rubella virus Day 40-45
  1. 1 Prepare RV inoculum for organoids
  2. 2 Apply RV inoculum to organoids
  3. 3 Replace inoculum with fresh media
  4. 4 Resume shaker culture
  5. 5 Prepare controls
  6. 6 Harvest organoids for short-term analysis
  7. 7 Continue culture for long-term analysis
Immunofluorescence staining of 2D cultures Day 3-4 post-infection
  1. 1 Fix cultures in paraformaldehyde
  2. 2 Wash with PBS
  3. 3 Block and permeabilize
  4. 4 Incubate with primary antibodies
  5. 5 Wash with wash buffer
  6. 6 Incubate with secondary antibodies
  7. 7 Final wash and imaging preparation
  8. 8 Image cultures
Immunofluorescence staining of brain slices and organoids Day 3-4 post-infection
  1. 1 Fix cortical slices
  2. 2 Fix and prepare organoids
  3. 3 Cryoprotect organoids
  4. 4 Prepare cryopreserved organoid sections
  5. 5 Block and permeabilize tissue sections
  6. 6 Incubate with primary antibodies overnight
  7. 7 Wash with wash buffer
  8. 8 Incubate with secondary antibodies overnight
  9. 9 Final wash and mounting
  10. 10 Image tissue sections
Viral titer quantification Variable (at designated timepoints)
  1. 1 Collect media supernatants
  2. 2 Clarify supernatants
  3. 3 Flash freeze samples
  4. 4 Prepare Vero cells for titering
  5. 5 Perform immunofluorescent titering assay
  6. 6 Calculate viral titer
Single-cell RNA sequencing sample preparation Day 3-4 post-infection
  1. 1 Dissociate organoids
  2. 2 Enzymatic digestion
  3. 3 Mechanical trituration
  4. 4 Filter cell suspension
  5. 5 Pellet cells
  6. 6 Wash cells
  7. 7 Resuspend cells for MULTI-seq barcoding
  8. 8 Prepare MULTI-seq barcodes
  9. 9 Add co-anchor
  10. 10 Final wash
  11. 11 Count cells and assess viability
10x Genomics single-cell capture Day 3-4 post-infection
  1. 1 Prepare 10x Genomics reagents
  2. 2 Load cells onto Chromium chips
  3. 3 Run Chromium controller
  4. 4 Perform reverse transcription
  5. 5 Recover cDNA
  6. 6 Amplify cDNA
  7. 7 Recover MULTI-seq barcodes
  8. 8 Continue standard 10x protocol with endogenous cDNA
  9. 9 Generate sequencing libraries
  10. 10 Sequence libraries
MULTI-seq barcode library preparation and sequencing Day 3-4 post-infection
  1. 1 Transfer barcode fraction to fresh tube
  2. 2 SPRI purification of barcode fraction
  3. 3 Recover barcode DNA
  4. 4 PCR amplify barcode fraction
  5. 5 Purify PCR products
  6. 6 Quality control of barcode libraries
  7. 7 Sequence barcode libraries
Single-cell RNA sequencing data analysis Post-sequencing
  1. 1 Process BCL files with CellRanger
  2. 2 Demultiplex samples with deMULTIplex
  3. 3 Detect and remove doublets with Solo
  4. 4 Apply QC filters
  5. 5 Remove ribosomal genes and pseudogenes
  6. 6 Perform batch correction
  7. 7 Normalize and scale expression data
  8. 8 Perform dimensionality reduction
  9. 9 Cluster cells with Leiden clustering
  10. 10 Annotate cell clusters
  11. 11 Identify differentially expressed genes
  12. 12 Filter marker genes
  13. 13 Generate visualization plots
  14. 14 Calculate Pearson correlation

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Popova et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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