Scripts to reproduce the snRNA-seq analysis of Populus trichocarpa stem

Introduction

This repository contains the source code necessary to reproduce the results described in the manuscript "Single-cell genomics of Populus stem reveals distinct cell population and developmental tracing of cambial derivatives"

Clustered .rds files necessary as inputs for analysis steps below can be downloaded from Figshare: https://doi.org/10.6084/m9.figshare.28392170.v1

The clustering of the snRNA-seq data was performed on Asc-Seurat v2.1.

The Protoplast single-cell data utilized for the comparative analysis from the article titled "Transcriptional landscape of highly lignified poplar stems at single-cell resolution" Genome Biol 22, 319 (2021). https://doi.org/10.1186/s13059-021-02537-2, was downloaded from NGDC BioProject Accession number PRJCA005543 (https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA005543). Data from "Response to: “Merit of integrating in situ transcriptomics and anatomical information for cell annotation and lineage construction in single-cell analyses of Populus”" Genome Biol 25, 86 (2024). https://doi.org/10.1186/s13059-024-03228-4 was downloaded from Zenodo - https://doi.org/10.5281/zenodo.10691111

NOTE: These steps requires the installation of dynverse, as shown at https://dynverse.org/users/1-installation/. In addition, Docker must be installed and running since dynverse requires it.

The .R code " SessionInfo.R" can be utilized to view and install all necessary packages and their respective versions utilized in this analyis.

Integration of SnRNA-seq Populus_trichocarpa stem reps 1 and 2

On Asc-Seurat, perform the integration using the file "Configuration_file_for_integration_analysis_SnRNAseq" as input and the default parameters of Asc-Seurat.

• Output: Integrated_dataSnRNAseqSTEMFinalclustering.rds

Once the integrated data is generated, it is possible to infer the developmental trajectories. Data should be reclustered according to the clustering guide in additional file 9.

• Output: XylemTrajectorysubsetNoC1.rds

Trajectory Analysis with Tradeseq was then carried out using the following code:

• trajectory_inference_tradeseq.R

Input file:

• HighresXyside.rds
• Stem_Tradeseq_Xylem.rds
  • Outputs:
  • EarlyDETest_Knots5,6FDRcorrected.csv
  • Tradeseq_diffEndTest_FDR.csv
  • 1.22.25_LINEAGE1_DEGs_tradeseq.csv
  • 1.22.25_DEGs_within_traj_of_lineage_2.csv
  • Tradeseq_association_test_output.xlsx

Protoplast and Nuclei Single-cell Comparison study

Protoplast Data was downloaded and processed on CellRanger using the following scripts:

• Download_protoplastData.sh
• PopCellRanger_Proto_Xylem2_counts.sh
• PopCellRanger_Proto_Phloem1to8.sh
• PopCellRanger_Proto_Xylem3to8_counts.sh

After protoplast data is downloaded and processed, using Asc-Seurat, perform the integration using the file "configuration_file_for_integration_analysisComparison.csv" as input and the default parameters of Asc-Seurat.

The following code was utilized to generate the plots for Figure1: Figure1DotplotCodeKnownMarkers.R