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Sample GSM8379684

Query DataSets for GSM8379684

Status

Public on Nov 27, 2024

Title

Control, repl3

Sample type

SRA

Source name

brain

Organism

Mus musculus

Characteristics

tissue: brain
cell type: microglia
genotype: GFP-M::Cx3cr1-CreERT2fl/fl::Rosa26-tdTomatotg/tg
treatment: control

Treatment protocol

Mice were fed with BLZ-945 (MedChemExpress, HY-12768) at a dose of 200 mg/kg b.w. daily in 100 µl PB (Peanut butter, GoOn, Santé); controls received orally PB. BLZ-945 was delivered for 14 or 21 days to establish the kinetics of depletion; repopulation was allowed for 7 days without any treatment.

Extracted molecule

polyA RNA

Extraction protocol

Mice were sacrificed and subsequently brains were collected, placed in cold HBSS (without Ca2+ and Mg2+), and transferred into 30 % sucrose (w/v) in PBS for 48 h and frozen in Tissue Freezing Medium (Leica) at -80°C. The tissue was minced, transferred onto c-tubes containing 1,950 µl DMEM, and supplemented with 50 µl of Deoxyribonuclease I (Sigma Aldrich). Dissociation was performed with the MACS dissociator with heaters (Militenyi Biotec) for 22 minutes and later the samples were passed through 70 nm and 40 nm strainers into 50 ml cold HBSS with ions to stop the enzymatic reaction (EasyStrainer™, BioOne).
For flow cytometry, cells were suspended in the Stain Buffer (BD Pharmingen) with anti-mouse CD16/CD32 Fc Block™ 1:200 (BD Pharmigen) for 10 min. Next, anti-mouse CD11b antibody 1:250 (M1/70, BD Pharmigen) was added and cells were incubated for 20 min at 4 °C, then washed with Stain Buffer. Cells were sorted using purity precision mode on FACS Aria (BD FACSAria Cell sorter BD-Biosciences), into 200 μL of sterile PBS.
For RNA isolation, sorted CD11b+ cells were stored in the -80°C freezer. The samples were thawed on ice and total RNA was isolated with the RNAeasy Plus Mini kit (Qiagen) according to the manual. RNA concentration was immediately determined using a spectrophotometer.
For bulk RNA-seq strand-specific polyA-enriched RNA libraries were prepared using the KAPA Stranded mRNA Sample Preparation Kit according to the manufacturer’s protocol (Kapa Biosystems, MA, USA). Library evaluation was done with Agilent 2100 Bioanalyzer using the Agilent DNA High Sensitivity chip (Agilent Technologies, Ltd.)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

We used rna-seq-star-deseq2 workbench v 2.0.0.
Sequence reads were trimmed using for adaptor sequence/ low quality reads using Cutadapt 3.0.
Trimmed sequence reads were mapped to GRCm38/mm10 genome assembly using STAR v. 2.7.10b
Read count extraction and normalization were done using edgeR v4.2.0.
Assembly: mm10
Supplementary files format and content: all.csv

Submission date

Jul 05, 2024

Last update date

Nov 27, 2024

Contact name

Patrycja Rosa

E-mail(s)

[email protected]

Organization name

Nencki Institute of Experimental Biology, Polish Academy of Sciences

Lab

Laboratory of Molecular Neurobiology