Page 14 - EJMO-9-2

Eurasian Journal of
Medicine and Oncology Single-cell sequencing for lung cancer

a uniquely barcoded gel bead. After cell lysis, mRNA binds mechanistic understanding of how epigenetic markers are
to oligo(dT) primers and undergoes reverse transcription established, maintained, and interpreted across different
(RT), incorporating cell barcodes and unique molecular cellular contexts. 32
identifiers (UMIs). The reaction droplets are then broken, The advent of single-cell multi-omics approaches
and the cDNA is polymerase chain reaction (PCR)- has significantly enhanced our understanding of cancer
amplified into sequencing-ready libraries. Using Illumina biology, including diseases like lung cancer. These
sequencing technology, the system captures barcodes, approaches integrate different molecular layers, such as the
UMIs, and mRNA sequences. Bioinformatics tools process genome and transcriptome, transcriptome and epigenome,
the data for single-cell transcriptomic profiling and gene and transcriptome and proteome, among others. In
expression analyses. scRNA-seq offers the analysis of single-cell genomics-plus-transcriptomics multi-omics
both coding and non-coding transcripts, allowing for technologies, genome and transcriptome sequencing
the discovery of novel gene isoforms, alternative splicing (G&T-seq) enables the parallel analysis of mRNA and
events, and non-coding genes that may contribute to 33
oncogenesis. 28,29 genomic DNA (gDNA) from the same single cell. After
cell lysis, polyadenylated mRNA is selectively captured
With a deeper understanding of gene regulatory using magnetic beads coated with oligo(dT) primers,
mechanisms, epigenetic modifications have been effectively separating it from nuclear and mitochondrial
recognized as key contributors. These modifications DNA. The mRNA is then reverse transcribed using a Smart-
include DNA base modifications and chromatin seq2-based protocol, incorporating template switching
structural changes. At present, a wide range of sequencing for full-length cDNA synthesis and PCR amplification.
technologies can measure chromatin SVs in epigenetics, This approach supports both short-read and long-read
from local to genome-wide levels. For example, a single- sequencing, with the latter being particularly useful
cell assay for transposase-accessible chromatin sequencing for transcript isoform detection. Meanwhile, gDNA is
(scATAC-seq) identifies open chromatin regions at the amplified using either multiple displacement amplification,
single-cell level using Tn5 transposase, which inserts which provides uniform coverage with minimal bias,
sequencing adapters into accessible chromatin. The or displacement preamplification followed by PCR,
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development of bulk ATAC-seq has resulted in three which enhances genome-wide coverage for downstream
distinct scATAC-seq strategies, including microfluidics- sequencing. By integrating transcriptomic and genomic
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based methods, split-and-pool combinatorial indexing, data from the same cell, G&T-seq facilitates the study of
and droplet-based procedures. Single-cell isolation is genotype-phenotype relationships, cellular heterogeneity,
achieved through microfluidics or droplet sorting, with and clonal evolution. Other single-cell genomics-plus-
barcoding used to distinguish DNA fragments. After transcriptomics multi-omics technologies include direct
amplification, library construction, and high-throughput RNA and DNA sequencing (DR-seq), targeted genomics
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sequencing, sequencing reads are mapped to a reference and transcriptomics sequencing (TARGET-seq), and
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genome to identify chromatin accessibility, classify cell single-cell one-tube sequencing (scONE-seq). These
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types, and infer gene regulatory networks. scATAC- technologies are particularly valuable in cancer research,
seq is widely applied to fresh, frozen, and fixed tissues, helping to uncover genomic instability and transcriptional
providing valuable insights into chromatin dynamics, dysregulation in diseases such as lung cancer.
cellular heterogeneity, and gene regulation. Single-cell
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epigenomic methods offer several significant advantages According to the central dogma, the main steps of gene
in studying complex biological systems. First, they enable expression include transcription and protein synthesis.
the analysis of the epigenome in rare or difficult-to-isolate However, gene expression also involves complex post-
cell populations, which are often inaccessible to traditional transcriptional and translational regulatory mechanisms.
bulk analysis techniques. Second, these methods facilitate Proteomics focuses on studying various properties of
the characterization of heterogeneous cell populations proteins, including their interactions, post-translational
by allowing for the classification of individual cells into modifications, expression, and regulation. It examines
known types or the discovery of novel subpopulations protein changes within an organism in a holistic manner to
with distinct epigenomic profiles. This approach enhances explain individual physiological and metabolic activities.
our understanding of cellular diversity and function The most significant distinction between proteomics
within tissues. Finally, single-cell epigenomics provides and traditional protein studies is that proteomics does
insights into the long-range correlations and dynamics not focus on studying one or a few proteins in isolation.
of epigenetic modifications across cell populations. Instead, it systematically investigates the overall response of
By analyzing these dynamics, researchers can gain a proteins, their expression, and interactions under different

Volume 9 Issue 2 (2025) 6 doi: 10.36922/ejmo.6883