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Sample GSM8242431

Query DataSets for GSM8242431

Status

Public on May 10, 2024

Title

Mouse-Rat library 2

Sample type

SRA

Source name

Connective tissue, forebrain, cord blood

Organism

Mus musculus x Rattus norvegicus

Characteristics

tissue: Connective tissue, forebrain, cord blood
mouse strain: CD1
rat strain: Wistar
genotype: Mouse TdT+ ESC

Treatment protocol

Embryonic day (E) 15.25 mouse-rat and E13.5 rat-mouse chimeras were dissected to harvest forebrain and connective tissue. In brief, chimeric fetuses were collected from either E15.25 rat or E13.5 mouse pregnant mothers and forebrain and connective tissues were harvested from those chimeric fetuses. Both forebrains and connective tissues were dissociated in the solution containing 2mg/ml Collagenase/Dispase (Roche, Basel, Switzerland; 10269638001) in Hanks' Balanced Salt solution. After 30-60 min incubation at 37 degrees, 10% fetal bovine serum (FBS) in PBS solution was added into the tissue solution to inactivate the enzymes. All the dissociated cells were then filtered and used for the subsequent experiment. Dissociated cells derived from forebrain and connective tissue were stained with APC-anti-mouse CD45 antibody (Biolegend, San Diego, CA; 103112), PE-Cy7-anti rat CD45 (Biolegend: 202214). Donor chimerism was analyzed by detecting CD45 negative, tdTomato or GFP-expressing cells (detailed in Table S2). Both donor and host cells in the forebrain and connective tissue of chimeric fetuses were sorted using a FACS Aria II (BD, Franklin Lakes, NJ).

Growth protocol

In brief, morula-stage embryos were obtained by uterus perfusion from superovulated CD1 mice at 2.5 days postcoitum (dpc). Morula-stage embryos were cultured in KSOM-AA medium (CytoSpring, Mountain View, CA; K0101) for 1 day and developed to blastocyst-stage embryos. Wild-type rat blastocysts were obtained by uterus perfusion from female rats at 4.5 dpc and cultured in Rat KSOM medium. For micromanipulation, mouse or rat ESCs were trypsinized and suspended in mouse or rat ESC culture medium. A piezo-driven micromanipulator (Prime Tech, Tsuchiura, Japan) was used to pierce the zona pellucida and trophectoderm under microscopy and 5–7 mouse or rat ESCs were introduced into blastocyst cavities near the inner cell mass. After blastocyst injection, embryos were cultured for 1–2 hours. Mouse blastocysts were then transferred into uteri of pseudopregnant recipient CD1 female mice at 2.5 dpc. Rat blastocysts were then transferred into uteri of pseudopregnant recipient Wistar female rats at 3.5 dpc. Table S1 shows results of the cell injection.

Extracted molecule

polyA RNA

Extraction protocol

Library preparation was done using the 10x v3.1 3' library preparation kit according to the manufacturer's instructions

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

All fastq files were aligned to the mouse (GRCm39, from ENSEMBL v109), rat (mRatBN7.2, from ENSEMBL v109), and a concatenated mouse and rat genome using CellRanger v7.1.048. For mapping, we retained protein-coding genes, lncRNA, antisense transcripts, snoRNA, snRNA, miRNA, and scaRNA. The resulting count matrices were read into scanpy and the proportion of aligned reads to each genome in the concatenated genome was used to determine the species of origin of each cell. A cell was assigned to mouse if greater than or equal to 70% of aligned reads aligned to the mouse genome, assigned to rat if greater than or equal to 70% of aligned reads aligned to the rat genome, and discarded as a doublet otherwise. For all subsequent steps, we only used the mouse-aligned-only counts for mouse cells and the rat-aligned-only counts for rat cells. We then removed all genes on sex chromosomes, restricted to mouse-rat one-to-one orthologs (defined using data from ENSEMBL), and performed standard pre-processing steps to remove low quality cells. For the latter, we performed standard filtering and removed cells with greater than 15% of counts coming from mitochondrial reads and cells with n_genes_by_counts (a commonly used quality control metric) greater than 7500. Next, we converted to the log normalized counts, identified highly variable genes, computed principal components, used the Python implementation of harmony to integrate the mouse and rat cells, and then found nearest neighbors. We then clustered the cells using the Leiden algorithm with resolution equal to 0.151. This first round of clustering split cells into neuronal, connective tissue, and hematopoietic lineages and then subsequent subclustering was done (see manuscript for details).
Assembly: mouse (GRCm39, from ENSEMBL v109), rat (mRatBN7.2, from ENSEMBL v109)
Supplementary files format and content: H5ad files were generated with Scanpy. All_Cells_Start.h5ad contains the raw counts from all the cells that passed quality control. Central_Nervous_System_Subclustered.h5ad contains normalized counts (see preprint for normalization details) and cell type information for central nervous system cell types. GABAergic_Forebrain.h5ad contains normalized counts and cell type information for forebrain GABAergic lineage cells from Central_Nervous_System_Subclustered.h5ad that were further subclustered. Chondrocyte_Subclustered.h5ad contains normalized counts and cell type information for chondrocyte lineage subtypes. Mesenchymal_Subclustered.h5ad contains normalized counts and cell type information for chondrocyte lineage subtypes.

Submission date

Apr 30, 2024

Last update date

May 10, 2024

Contact name

Hunter B. Fraser

E-mail(s)

hbfraser@stanford.edu

Organization name

Stanford University

Street address

371 Jane Stanford Way