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Sample GSM4589119

Query DataSets for GSM4589119

Status

Public on Jun 04, 2020

Title

Pb.M2

Sample type

SRA

Source name

hippocampal cells

Organism

Mus musculus

Characteristics

Sex: Male
treatment: Pb

Treatment protocol

Post-pubertal virgin a/a females (~5 weeks old) were randomized into exposure groups: control or drinking water containing 32ppm Pb-acetate. This dose and route of exposure was designed to be relevant to human perinatal exposure. After an initial two weeks of exposure, females were mated with a/a males. Exposure through drinking water continued through gestation and lactation. After weaning, 1 male and 1 female wildtype (a/a) offspring per litter were maintained on Pb-free drinking water. At 5 months, mice were sacrificed for experimental analyses. All animals had access to food and water ad libitum throughout the experiment, were housed in polycarbonate-free cages, and were maintained in accordance with the guidelines set by the Institute of Laboratory Animal Resources.

Extracted molecule

total RNA

Extraction protocol

During sacrifice, mice were perfused with a saline solution. The hippocampal region of the brain was isolated, and then dissociated into a viable single cell suspension using the Adult Mouse and Rat Brain Dissociation kit (Miltenyi) using a gentleMACS Octo Dissociator with Heaters automated tissue dissociation instrument. Cells were cryopreserved using Recovery Cell Culture Freezing Medium (Thermo Fisher). Viability and cell concentrations were quantified upon thawing for single cell analysis using a Luna FL Automated Cell Counter (Logos) by co-quantification of fluorescence from acridine orange and propidium iodide dyes.
Single cells were processed for high throughput RNA sequencing using the Chromium (10x Genomics) instrument, attempting to capture approximately 3,000-5,000 cells per sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Raw single cell RNA-sequencing data were processed using the CellRanger (10x Genomics) analysis pipeline. mkfastq was used to demultiplex the raw Illumina BCL files into fastq format. count was used to align the reads to the mouse reference genome (mm10), filter reads, count barcodes, and count UMIs. The output from count yields digital gene expression (DGE) matrices for each sample that contain the UMI counts per gene, per cell barcode. DGEs were loaded into the single cell transcriptomic data analysis suite Seurat, then normalized.
We excluded droplets with fewer than 1,000 expressed genes and we excluded genes that were measured in fewer than three cells, leaving a total of 5,258 cells and 17,143 genes expressed across the 8 samples.
Genome_build: mm10
Supplementary_files_format_and_content: matrix table with gene counts after filtering droplets and genes

Submission date

Jun 03, 2020

Last update date

Jun 04, 2020

Contact name

Kelly Bakulski

E-mail(s)

bakulski@umich.edu

Organization name

University of Michigan

Department

Department of Epidemiology