Recent technological advancements have enabled spatially resolved transcriptomic profiling but at multi-cellular pixel resolution, thereby hindering the identification of cell-type-specific spatial patterns and gene expression variation. To address this challenge, we develop STdeconvolve as a reference-free approach to deconvolve underlying cell types comprising such multi-cellular pixel resolution spatial transcriptomics (ST) datasets. Using simulated as well as real ST datasets from diverse spatial transcriptomics technologies comprising a variety of spatial resolutions such as Spatial Transcriptomics, 10X Visium, DBiT-seq, and Slide-seq, we show that STdeconvolve can effectively recover cell-type transcriptional profiles and their proportional representation within pixels without reliance on external single-cell transcriptomics references. **STdeconvolve** provides comparable performance to existing reference-based methods when suitable single-cell references are available, as well as potentially superior performance when suitable single-cell references are not available. STdeconvolve is available as an open-source R software package with the source code available at https://github.com/JEFworks-Lab/STdeconvolve .

In the realm of transcriptional dynamics, understanding the intricate interplay of regulatory proteins is crucial for deciphering processes ranging from normal development to disease progression. However, traditional RNA velocity methods often overlook the underlying regulatory drivers of gene expression changes over time. This gap in knowledge hinders our ability to unravel the mechanistic intricacies of these dynamic processes. scKINETICs (Key regulatory Interaction NETwork for Inferring Cell Speed) (Burdziak et al, 2023) offers a dynamic model for gene expression changes that simultaneously learns per-cell transcriptional velocities and a governing gene regulatory network. By employing an expectation-maximization approach, scKINETICS quantifies the impact of each regulatory element on its target genes, incorporating insights from epigenetic data, gene-gene coexpression patterns and constraints dictated by the phenotypic manifold.

Single-cell RNA sequencing (scRNA-seq) data have allowed us to investigate cellular heterogeneity and the kinetics of a biological process. Some studies need to understand how cells change state, and corresponding genes during the process, but it is challenging to track the cell development in scRNA-seq protocols. Therefore, a variety of statistical and computational methods have been proposed for lineage inference (or pseudotemporal ordering) to reconstruct the states of cells according to the developmental process from the measured snapshot data. Specifically, lineage refers to an ordered transition of cellular states, where individual cells represent points along. pseudotime is a one-dimensional variable representing each cell’s transcriptional progression toward the terminal state. Slingshot which is one of the methods suggested for lineage reconstruction and pseudotime inference from single-cell gene expression data. In this notebook, we will illustrate an example workflow for cell lineage and pseudotime inference using Slingshot. The notebook is inspired by Slingshot's vignette and modified to demonstrate how the tool works on BioTuring's platform.