Development of single-cell transcriptomic atlas of human plasmacytoid dendritic cells from people with HIV-1

Summary Many immunological treatment strategies for reducing the HIV-1 reservoir and enhancing adaptive immunity aim at activating the human plasmacytoid dendritic cells (pDCs). Here, we present a protocol for pDC enrichment, single-cell analysis, and development of a pDC transcriptomic database from healthy individuals and people with HIV-1 before and after Toll-like receptor 9 agonist treatment. For complete details on the use and execution of this protocol, please refer to Cham et al.1


Single cell library preparation and sequencing
Timing: 2-3 days 5. Sc-RNA library construction using the Chromium Next GEM Single Cell 3 0 Reagent Kits v3. 1

Development of pDC transcriptomic database
Timing: 1-2 days 10.Develop dataset of pDCs from healthy individual's vs. PWH (https://dreamapp.biomed.au.dk/HIV_pDC/control_HIV_pDC/). 11.Develop dataset of pDCs from PWH before and after MGN1703 treatment (https://dreamapp.biomed.au.dk/HIV_pDC/HIV_TLR9_pDC). Note: perform flow cytometry of PBMCs before and after dead cells removal to ensure that the dead cell removal is successful.This can be done by staining with viability dye (Near-IR) for 20 min at 4 C.

Human pDC enrichment
Timing: 2 h This section provides the requirements that are necessary to enrich human pDCs from PBMCs.f.Again, wash cells by adding at least 5 mL of buffer, centrifuge at 300 3 g for 10 min and gently aspirate supernatant.g.Suspend cells in 500 mL of buffer and proceed to magnetic separation.h.Put cell suspension in ice and or 4 C.Meanwhile, place LD Column in the MACS Separator and rinse with 2 mL of buffer (this step should be done during the last incubation).i.Put the cell suspension cell onto the column, collect unlabeled cells that pass through and wash column with 2 mL of buffer.4. Flow cytometry validation using pDC surface markers (CD303 and CD123).

Timing: 2-3 days
This section provides the requirements that are necessary to perform single RNA-seq of enriched pDCs.The volume of certain reagents in preparing master Mix depends on the number of reactions (e.g., 13, 43 or 83) described in detail in the 103 genomic manual.5. Sc-RNA library construction using the Chromium Next GEM Single Cell 3 0 Reagent Kits v3.1.

Note:
Reagents and equipment's should be prepared while enriching pDCs and start the single cell library preparation immediately after enrichment.a. Prepare reaction mix and GEM loading.
i. Calculate the number of cells to be used and target cell recovery.
ii. Prepare master mix on ice.The master should include RT Reagent B, Template Switch Oligo, Reducing Agent B and RT Enzyme B.
Note: Pipette mix each reagent before adding to master mix.The volume for the number of reactions is well described in the 103 Genomic manual.
iii.Add 50% glycerol solution to each unused well on the Next GEM Chip G. Add 70 mL in row 1, 50 mL in row 2 and 45 mL in row 3. iv.Prepare master mix with pDCs suspension and add appropriate volume of nuclease-free water and mix.

Note:
The volume of cell suspension and nuclease-free water depend on the cell concentration and well described in the 103 Genomic manual.
v. Load 70 mL of pDCs + master mix in the row 1 of the Next GEM Chip G. i. Prepare cDNA Amplification reaction Mix using Amp mix and Feature cDNA primer 3.The volume depends on the number of reactions (e.g., 13, 43 or 83) described in detail in the 103 genomic manual.

Note
ii. Add 65 mL cDNA Amplification Reaction Mix to 35 mL sample, pipette mix and centrifuge briefly.iii.Incubate in a thermal cycler following protocol recommended in the 103 genomic manual.

GEM-RT Incubation
Steps Temperature Time i. Use only 10 mL cDNA sample.This is enough to generate 3 0 Gene expression library.
Note: The remaining 30 mL cDNA sample should be stored in À20 C.
ii. Prepare fragmentation mix using fragmentation buffer and fragmentation enzyme.The volume depends on the number of reactions (e.g., 13, 43 or 83) described in detail in the 103 genomic manual.
iii.Add 25 mL Buffer EB to each sample, then add 15 mL Fragmentation mix, pipette mix and centrifuge briefly.iv.Transfer into thermal cycler following incubation protocol which is described in detail in the 103 genomic manual.i. Add 50 mL Amp mix to new tubes containing the transfer supernatant.Then add 20 mL of an individual Index TT Set A to each sample, pipette mix and centrifuge briefly.
Note: Record the well ID of the Index TT Set A.
ii. Incubate the sample in a thermal cycler following incubation protocol which is described in detail in the 103 genomic manual.
iii.Once thermal cycler completes, add 60 mL SPRIselect reagent, pipette mix and incubate for 5 min at room temperature.iv.Place on magnet (high) until the solution is clear and transferred 150 mL supernatant to new tubes.v. Add 20 mL of SPRIselect to the new tubes containing the transferred supernatant, pipette mix and incubate for 5 min at room temperature.vi.Place on magnet (high) until the solution is clear and remove 165 mL supernatant and add 200 mL ethanol to the pellet for 30 s. Remove ethanol and repeat wash twice.vii.Place in magnet (low), remove excess ethanol and add 35.5 mL Buffer EB, pipette mix and incubate for 2 min at room temperature.viii.Place magnet (low) until the solution is clear, and transfer 35 mL to new tubes.

Note:
The transferred supernatant is used for quality control and sequencing.The remaining sample should be stored in À20 C. g.Post library construction quality control.
i. Run 1 mL sample at 1:10 dilution on an Agilent Bioanalyzer High Sensitivity chip.
Note: Determine the average fragment size from the Bioanalyzer trace.This will be used as the insert size for library quantification.

Sequencing.
Note: Our samples were sent to the Department of Molecular Medicine at the Aarhus University Hospital for sequencing.Single-cell barcoded cDNA libraries were sequenced on an Illumina NovaSeq 6000 system (100-cycle cartridge) with a sequencing depth of at least 50,000 reads per cell.You can also send your samples for sequencing to other companies.

Analysis of sc-RNA data
Timing: 3-4 days Sample Index PCR (# of cycle is 10-12) Steps Temperature Time In this section, we describe how to analyze sc-RNA-seq data.The protocol starts by sequence alignment of raw scRNA-seq fastq files from each sample generate the cell-genes count matrices.Importing filtered feature-barcode matrices, excluding bad-quality cells, non-expressed genes and doublets.Cluster analysis, annotation and visualization and selection of pDC clusters for in-depth characterization.
7. Alignment, Quality control of RNA-seq data.a. Individual raw scRNA-seq fastq files from each sample should be aligned against the human reference genome (GRCh38) through the cell ranger count pipeline (Cell Ranger version 3.1.0,103 Genomics Technology), which will generate the cell-genes count matrices.b.Use R studio and the Seurat package (version 4.0.3) to perform a clean-up and quality control based on cellular expression of mitochondrial genes (cells with a percentage of mitochondrial genes >5% were discarded).Use DoubletFinder algorithm to remove doublets.Note: Since pDC does not yield 100% purity and as shown in that there is contamination from other immune cells in the dataset, it's important to select the pDC cluster for further in-depth analysis.

QUANTIFICATION AND STATISTICAL ANALYSIS
Differential expression genes (DEGs) were analyzed using three tests, Wilcoxon-ranked sum test, t-test and t-test overestimated variance.DEGs were computed using the 'FindMarker' function of Seurat and the probability values were estimated with respect to all other clusters within each dataset.

LIMITATIONS
Human pDCs only make up 0.1%-0.5% of all PBMCs and large blood volumes are required to obtain enough cells for the experiments.Enrichment of pDCs contains a lot of infiltrating immune cells.Time of sample collection is important to target interferon genes.Using cryopreserved PBMCs for pDC experiment is not very ideal, rather freshly isolated PBMCs is preferable.

TROUBLESHOOTING Problem 1
Cryopreserved PBMCs yield poor cell viability.

Potential solution
Use freshly isolated PBMC.

Problem 2
Lot of infiltrating immune cells in the enriched pDC.

Potential solution
Perform cell sorting instead of cell enrichment.

Problem 3
Impossible or hard to perform surface protein analysis along with gene expression on pDCs.

Potential solution
Ensure that enough PBMCs are available for pDC isolation.

Problem 4
Quality control of raw pDC sequence data has high mitochondria markers.

Potential solution
Perform single cell RNA library preparation immediately after isolation.

Lead contact
Individual participant data cannot be made available due to EU Data Protection Regulations (GDPR).
A limited and completely anonymized version of the dataset can be obtained upon request.Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact (Ole S. Søgaard): olesoega@rm.dk.

Data and code availability
Single cell RNA-seq raw and processed data have been deposited at Gene Expression Omnibus (GEO accession GSE228078) https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE228078.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Note:
It is recommended but not required to perform flow cytometry of enriched cells to check the purity.a. Block non-specific binding and stain cells with pDC surface markers CD303 and CD123 antibodies for 30 min at 4 C. b.Perform flow cytometry and analyze pDC purity.Note: pDCs are fragile and should always be stored in ice or at 4 C. Start the single cell library preparation immediately after enrichment.
You can stop and store sample at 4 C for up to 72 h or À20 C for %1 week or proceed to the next step.iv.Vortex to resuspend the SPRIselect reagent and add 60 mL SPRIselect reagent to each sample.v. Pipette mix and incubate for 5 min at room temperature.Then place on a magnet (high) until it's clear.Remove supernatant from pellet.CRITICAL: DO NOT discard the pellet vi.Add 200 mL 80% ethanol to the pellet for 30 s, remove the ethanol and repeat wash.vii.Centrifuge briefly and place on magnet (low) for 2 min.Remove remaining ethanol and air dry.viii.Add 40.5 mL Buffer EB, mix and incubate for 2 min at room temperature.ix.Place on magnet (high) until the solution is clear.x.Transfer 40 mL sample to a new tube strip.xi.Run 1 mL of sample, diluted 1:10 on an Agilent Bioanalyzer High Sensitivity chip.CRITICAL: Please ensure that each sample yields a good cDNA concentration before proceeding.d.Post Fragmentation, End Repair & A-tailing Double Sided Size Selection.

5
Contamination of other immune clusters.Potential solution Perform cluster selection and cleanup.
Thaw PBMCs and filter cells.2. Removing dead cells using EasySep Dead Cell Removal kit.
. a. Master mix preparation and GEM loading.b.GEM generation and cleanup.c. cDNA amplification, cleanup, and quantification.d.Fragmentation, Post Fragmentation, End Repair & A-tailing Double Sided Size Selection.
e. Adaptor Ligation and Post Ligation Cleanup.f.Sample Index PCR and Post Sample Index PCR Double Sided Size Selection.g.Post library construction and quality control.6. Sequencing.

TABLE REAGENT
(Continued on next page) 3. pDC enrichment using human plasmacytoid dendritic cells isolation kit II.a.Cells suspension obtained after dead cell removal should be kept in ice or 4 C. b.Centrifuge the cell suspension at 300 3 g for 10 min and gently aspirate supernatant.c.Suspend cell pellet in 400 mL of buffer, add 100 mL of the Non-PDC Biotin-Antibody Cocktail I, mix and incubate for 10 min in ice or 4 C. d.Wash cells by adding at least 5 mL of buffer, centrifuge at 300 3 g for 10 min and gently aspirate supernatant.e. Add 400 mL of buffer, add 100 mL of the Non-PDC MicroBead Cocktail II, mix and incubate for 15 min in ice or 4 C.
Note: During incubation, please prepare MACS Column and MACS Separator.It is recommended to place LD Column in the MACS Separator and rinse with 2 mL of buffer.
Remove the supernatant, add 300 mL 80% ethanol to the pellet while on the magnet and discard ethanol.Repeat ethanol wash and air dry.
viii.Prepare Elution Solution-I using buffer EB, 10% tween 20 and reducing agent B. The volume depends on the number of reactions (e.g., 13, 43 or 83) described in detail in the 103 genomic manual.ix.Add 35.5 mL Elution Solution I, pipette mix and incubate for 2 min at room temperature.x.Transfer 35 mL sample to a new tube strip.c. cDNA amplification, cleanup, and quantification.
Once the thermal cycler completes, vortex the SPRIselect and 30 mL to each sample and incubate for 5 min at room temperature.vi.Place samples in magnet (high) until the solution is clear and transfer 75 mL supernatant into new tubes.vii.Add 10 mL SPRIselect to the new tubes containing transferred supernatant and incubate for 5 min at room temperature.viii.Place samples in magnet (high) until the solution is clear and discard 80 mL supernatant.CRITICAL: Do not discard the bead, use tubes containing beads in the next step.ix.Add 125 mL ethanol to the pellet for 30 s, remove ethanol and repeat wash twice.Remove remaining ethanol and air dry.x.Add 50.5 mL Buffer EB to each sample, pipette mix and incubate for 2 min at room temperature.xi.Place samples in magnet (high) until the solution is clear and transfer 50 mL sample into new tubes.e.Adaptor Ligation and Post Ligation Cleanup..Place on magnet (high) until the solution is clear, then remove the supernatant.vi.Add 200 mL ethanol to pellet for 30 s, remove ethanol and repeat wash twice.Remove remaining ethanol and air dry.vii.Add 30.5 mL Buffer EB and incubate for 2 min at room temperature.Place on magnet (low) until the solution is clear and then transfer 30 mL supernatant to new tubes.f.Sample Index PCR and Post Sample Index PCR Double Sided Size Selection.
i. Prepare Adaptor Ligation mix using Ligation Buffer, DNA Ligase, and Adaptor Oligos.The volume depends on the number of reactions (e.g., 13, 43 or 83) described in detail in the 103 genomic manual.ii.Add 50 mL Adaptor Ligation mix to the 50 mL sample and pipette mix.iii.Transfer to thermal cycles following incubation protocol which is described in detail in the 103 genomic manual.iv.Once the thermal cycle completes, add 80 mL SPRIselect reagent to each sample, pipette mix and incubate for 5 min at room temperature.v Our protocol describes methods of human pDC enrichment, single cells RNA-seq analysis and development of human pDC transcriptomic database from healthy, PWH before and after TLR9 agonist (MGN1703) treatment.Following the step-by-step above, you can expect to successfully enrich enough live pDCs for your single cells experiment and how to construct a database for your single cell transcriptomic data.Development of pDC transcriptomic atlas helps easy understanding for non-computational researchers.