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Sample GSM2845484

Query DataSets for GSM2845484

Status

Public on Dec 01, 2017

Title

202

Sample type

SRA

Source name

Microglia

Organism

Mus musculus

Characteristics

strain: C57BL/6
tissue: Brain>Cerebellum
genotype: Wild Type
treatment: PBS

Extracted molecule

total RNA

Extraction protocol

All procedures were carried out at 4°C. Mice were perfused, cerebella were chopped and homogenized using a Dounce homogenizer in 2ml cold Medium A (HBSS+ 15mM HEPES+0.05% glucose+ 1:500 DnaseI), filtered through 100µm cell strainer, rinsed with 5ml Medium A and centrifuged at 340g for 5 mins. For myelin removal, the precipitate was resuspended in 25% standard isotonic percoll (25% Percoll in PBS, diluted with Medium A) and centrifuged at 950g for 20 mins. CD11b+Tmem119+ microglia (~20,000 per sample) were FACS sorted into 500µl RLT buffer (Qiagen) and stored on -80°C.
RNA was extracted from isolated microglia using RNeasy Plus Micro kit (Qiagen) and the quality assessed by Agilent 2100 Bioanalyzer (Stanford PAN facility). About 1ng RNA was converted to cDNA and amplified for 12 cycles using SMART-seq v4 Ultra Low input RNA kit for sequencing (Takara Bio USA, Inc.) according to manufacturer’s instructions. Amplified cDNA was then purified by immobilization on AMPure XP beads. Purified cDNA was normalized and tagmented for 5 mins using Nextera XT DNA library prep kit (Illumina). Unique indexes were then added to each sample, which were then amplified for 12 cycles. cDNA was purified using AMPure XP beads and quality assessed by Advanced Analytical Fragment Analyzer (Stanford PAN facility). Samples were then normalized and pooled together and sequenced on Illumina Novaseq 6000 (Novogene Corporation Inc.) to obtain 150bp paired-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

NormalizedCounts.csv

Data processing

The quality of fastq files was assessed using FASTQC (v 0.11.4)
Reads were trimmed to 75bp using fastX toolkit (v 0.0.14)
Trimmed reads were mapped to mouse mm9 reference genome using STAR (v 2.5.1b). Raw read counts were generated with STAR using the GeneCounts function.
Differential expression in RNA-Seq was analyzed using the R DESeq2 package
Differential expression in RNA-Seq was analyzed using the R DESeq2 package. Read counts were used as input and normalized using built-in algorithms in DESeq2.
Genome_build: mm9
Supplementary_files_format_and_content: comma separated value file of normalized read counts from DESeq2 software

Submission date

Nov 08, 2017

Last update date

May 15, 2019

Contact name

Liana Nicole Bonanno

Organization name

Stanford University

Department

Neurology

Lab

Tony Wyss-Coray