DOI: 10.5281/zenodo.15720855
title: Spatial Transcriptomics Reveals Expression Gradients in Developing Wheat Inflorescences at Cellular Resolution

description: This repository contains the transcriptomic datasets summarised in its associated publication. It contains zipped folders, with the following structure:

 

MERFISH results: 

The dataset is comprised of eight gzipped tar archives (2 genotypes by 4 timepoints), containing the outputs of the VizGen MERSCOPE platform, in combination with the results of CellPose2 cell segmentation.

The timepoints of inflorescence development used here are:

- Late Double Ridge stage (LDR, Waddington stage W2.5)

- Lemma Primordium stage (LP, Waddington stage W3.25)

- Terminal Spikelet stage (TS, Waddington stage W4)

- Carpel Extension stage (CER, Waddington stage W5)

 

The genotypes described are:

- ‘WT’: Triticum aestivum, BC6 near-isogenic lines (NILs) in the UK background Paragon, carrying VRT-A2a allele.

- ‘POL’: Triticum aestivum, BC6 near-isogenic lines (NILs) in the UK background Paragon, carrying the VRT-A2b allele. 

 

The archive files are named to summarise the represented genotype/timepoint:

- MERFISH_Results_LDR_WT.tar.gz,

- MERFISH_Results_LDR_Pol.tar.gz,

- MERFISH_Results_LP_WT.tar.gz,

- MERFISH_Results_LP_POL.tar.gz,

- MERFISH_Results_TS_WT.tar.gz,

- MERFISH_Results_TS_POL.tar.gz,

- MERFISH_Results_CER_WT.tar.gz,

- MERFISH_Results_CER_Pol.tar.gz

 

Each archive files contains the following:

- The segmented Cellpose2 output for that particular genotype and timepoint for use within the MERSCOPE program developed by VizGen.

- A folder with the MERSCOPE output data; (i) a matrix with the detected transcript counts in csv format, (ii) a file detailing the transformation of microns to mosaic pixels in csv format, (iii) a tif image with the raw data output from the DAPI channel, and (iv) a tif image of the raw output of the polyT channel.

- A folder with the processed segmentation output; (i) a cell-by-gene expression matrix in csv format, (ii) a metadata file for the cells in csv format, (iii) the cellpose2 parquet data in microns, (iv) the cellpose2 parquet data in mosaic pixels, (v) a json file with the segmentation specifications, (vi) another folder containing the DAPI and polyT images after manual removal of seam lines.

The metadata information for each zipped file is summarised in file ‘MERSCOPE_samplepreparation_metadata.xlsx’

 

RNA-seq microdissection dataset: 

A zipped archive folder, ‘RNAseq.tar.gz’, containing:

- SampleMetadata_RNAseq_ImpulseDE2analysis.csv (sample metadata used in RNA-seq sample collection & sequencing)

- TPM_RNAseq_ImpulseDE2results.csv (RNA seq expression values (TPM: transcript per million) and results of ImpulseDE2 analysis)

For raw reads, see: NCBI, BioProject number PRJNA1201104

 

MERFISH QC analysis: 

A zipped archive folder, ‘QC.tar.gz’, containing:

- cellmetadata_genecounts_transcriptcounts.csv (a cell x gene matrix, containing information of cell id, cell size, cell coordinates, sample ID, number of genes, number of transcripts, and total counts per transcript id for 200 genes)

- blankcounts_persample.csv (total counts of blank probes detected per sample)

- transcriptcounts_genecounts_summarystatistics.csv (sample summary statistics of total counts per cell and gene counts per cell)

- Eight .h5ad files, containing the transcriptomic and cell metadata information for each sample after filtering out low quality cells and normalising counts, for use in further analysis with python-based tools.

- metadata_insilico_dissections.csv (metadata information on the in-silico ‘sections’ made in MERSCOPE Visualizer Tool)

- metadata_insilico_dissections.csv (Spearman’s Correlation Coefficients and significance values between ‘in silico’ sections and RNAseq TPM values)

- A folder of geometry files (.csv format) summarising the in silico ‘sections’

 

MERFISH Sample Integration and Clustering Analysis: 

A zipped archive folder, ‘SampleIntegration_Clustering.tar.gz’, containing:

- DomainAssignment_PerCell.csv (expression domain assignment of each cell id)

- VRT_A2a_A2b_leiden_200genes_res1.hdf5 (AnnData object containing metadata on domain assignment for each cell, to be imported to the MERSCOPE Visualizer tool for further exploration of domain assignments)

 

MERFISH Gene Enrichment Analysis: 

A zipped archive folder, ‘GeneEnrichmentAnalysis.tar.gz’, containing:

- LogRegScore_NormExpAvg_allsamples.csv (results of gene enrichment analysis, with the logistic regression scores, and normalised expression of each transcript per domain)

- topenrichedgenes_domainnames_annotated.xlsx (annotated domains and their significantly enriched genes)

- ABCDE_gene_table.xlsx (Naming conventions of genes in the ABCDE model across wheat, barley, maize, and rice)

- P1POL_W4_VRT_SEP_CoExpression.csv and P1WT_W4_VRT_SEP_CoExpression.csv (Tables on the rates of co-expression of VRT2 and SEP1-4 in two samples at stage W4)

- cellsinspike_W4_P1POL.csv and cellsinspike_W4_P1WT.csv (Table summarising cells included in the inflorescence of stage W4 inflorescence samples)

- Averaged_NormalisedCountsperCell_SRLRgroups.csv (Normalised transcript counts averaged within each spikelet ridge (SR) or leaf ridge (LR) grouping)

- PC1_loading_scores_SRLRgroups.csv (PC1 loading scores for analysis on spikelet ridges and leaf ridges)

- P1WT_LDR_cell_categories.csv (a table of cells incorporated into spikelet ridge and leaf ridge groupings)

- P1WT_LP_allcellsinspike.csv and P1WT_LDR_allcellsinspike.csv (tables summarising cells included in inflorescence of stage W2.5 and W3.25)