eBook: Advances in Cancer Immunology Research - 15

applied to current bulk RNA-seq workflows to generate
complementary protein data and more informative
datasets overall.
Materials and Methods
Sample processing and cytokine detection
Human PBMCs were isolated from healthy donors
and stimulated with phytohemagglutinin (PMA) and
Ionomycin for 4 hours. An aliquot was also treated
with monensin to measure intracellular cytokines
using flow cytometry and secreted cytokines using
LEGENDplex™, following BioLegend protocols. Flow
Cytometry was performed using a BD LSRFortessa™,
and it was analyzed using FlowJo™.
Bulk Sequencing
To incorporate protein analysis to bulk RNA-seq, PBMCs
for BEN-seq were stained with 280 TotalSeq™
antibodies using BioLegend's bulk epitope and nucleic
acid sequencing (BEN-seq) Protocol.
RNA and antibody-derived tags (ADTs) fractions of
PBMCs were isolated using a column based whole
blood RNA isolation kit (pelleted cells protocol) with
proteinase K but without the DNAse step. RNA and
protein associated libraries (ADTs) were separated
using streptavidin-magnetic beads loaded with
complementary oligonucleotides using BioLegend's
BEN-seq protocol (Figure 3). The remaining RNA
fraction was processed using the Illumina TruSeq™
Stranded mRNA Library Prep kit with Illumina -
TruSeq™ RNA UD Indexes. Samples were sequenced
on the Illumina MiniSeq™ Sequencing System.
Single-Cell Sequencing
PBMCs for scRNA-seq were stained with 280 TotalSeq™
antibodies using BioLegend's TotalSeq™-A Antibodies
and Cell Hashing with 10x Single Cell 3' Reagent
Kit v3/3.1 Protocol. Cells were counted with a
Countess™ II FL Automated Cell Counter and loaded
into a 10x Genomics Chromium Controller. The Single
Cell 3' Reagent Kit v3.1 was used according to the
manufacturer's protocol for single-cell RNA (scRNA)
isolation. ADT libraries were generated in parallel
using BioLegend's TotalSeq™-A antibodies. Samples
were multiplexed using BioLegend's Cell Hashing
reagents, and sequenced on the Illumina NovaSeq™
6000 Sequencing System.
Data Analysis
Bulk RNA-seq raw reads were normalized using regularized
log as implemented by DESeq2. For scRNAseq
analysis, the number of unique molecular identifiers
(UMIs) mapped to each gene/barcode was
normalized using an arsinh (inverse hyperbolic sine)
transformation. Heatmaps were plotted using R's
ComplexHeatmap module.
BioLegend protocols, including BEN-seq, available at
biolegend.com/en-us/technical-protocols
Results
PBMCs from two donors were isolated and separated
into two groups. Cells from Group 1 underwent
a five-day Th1 polarization and Group 2 served as a
negative control. After the five-day polarization period,
cells were stained with a 280 antibody TotalSeq™-A
panel. Both groups were further divided into
subgroups for single-cell or bulk RNA-seq experiments.
BEN-seq was performed on a MiniSeq™ and
single-cell sequencing was performed on a NovaSeq™
6000 (Figure 1).
To verify the outcome of the Th1 polarization, intracellular
expression of the Th1 associated cytokines
IL-2, IFN-γ, and TNF-α was measured by flow cytometry
on polarized cell suspensions (Figure 2, dot
15
http://www.biolegend.com/en-us/technical-protocols
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