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Sample GSM8021374

Query DataSets for GSM8021374

Status

Public on Apr 16, 2024

Title

Sham_Mg_R1

Sample type

SRA

Source name

brain

Organism

Mus musculus

Characteristics

tissue: brain
celltype: Microglia (Mg)
genotype: wildtype
treatment: Sham control

Extracted molecule

total RNA

Extraction protocol

Following TBI experiments, animals were perfused with 4C DPBS and a 1 mm tissue block spanning the lesion sites of each mouse was isolated. To increase cell yield for RNA-seq analysis, two mouse brain tissue samples per genotype and timepoint were randomly pooled together for each biological replicate. Single cell suspensions were prepared using the adult brain dissociation kit (Miltenyi Biotec). Samples were then treated with Fc-block in DPBS supplemented with 0.2% BSA (Thermo Fischer Scientific) for 5 min at 4C, then incubated with primary antibodies for 30 min at 4C. The following primary antibodies were used at 1:200 dilution and purchased from Biolegend: CD11b (M1/70) conjugated to APC-Cy7 and CD45 (30-F11) conjugated to 488. Samples were incubated with Sytox blue live/dead stain for 10 min prior to sorting. FACS of 40,000 live microglia with CD45lowCD11b+ expression or 25,000 live infiltrating myeloid cells with CD45hiCD11b+ expression were sorted directly into tubes containing RLT plus lysis buffer (Qiagen) supplemented with 1% 2-mercaptoethanol and 0.25% reagent DX (Qiagen) using FACSAria II with BD FACSDiva v8 software.
cDNA libraries were generated using the Ovation RNA-seq System V2 low input kit following manufacturer’s instructions (NuGEN). Libraries were QC checked by KAPA qPCR (Roche) and Bioanalyzer DNA chip analysis (Agilent). All 21 libraries were equimolar pooled and sequenced on Hiseq4000 with paired-end 100 base pairs (Illumnia). The median sequencing depth was ~63 million reads per sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

Sham_Mg_R1

Data processing

Paired-end fastq files were processed using the open-access Nextflow RNA-seq pipeline with nextflow v20.12.0-edge in Singularity with default nf-core/rnaseq v3.0 parameters and packages.
Fastq files were mapped to GRCm38.mm10 genome. Gene analyses was performed in R v4.2.0 using DeSeq2 v1.36.0 on the salmon.merged.gene_counts_scaled file. Reads with fewer than 3 counts per gene across replicates were filtered out.
For differential gene analysis the results function in DeSeq2 was used with contrast to test between two genotypes and timepoints of interest. The data were then filtered for significance using abs(log2FC) > 1 and adjusted P value < 0.05 unless otherwise stated. Unbiased KEGG analysis was performed using clusterProfiler and enrichplot with default parameters and pvalueCutoff set to 0.1.
Assembly: mm10
Supplementary files format and content: txt file containing salmon processed data

Submission date

Jan 17, 2024

Last update date

Apr 16, 2024

Contact name

Katerina akassoglou

E-mail(s)

katerina.akassoglou@gladstone.ucsf.edu

Organization name

Gladstone Institutes

Street address

1650 Owens st