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Sample GSM3466727

Query DataSets for GSM3466727

Status

Public on Nov 14, 2018

Title

FB_RNA_rep2

Sample type

SRA

Source name

E15.5 forebrain dopaminergic neurons

Organism

Mus musculus

Characteristics

strain: Tg(Th-EGFP)DJ76Gsat x Swiss Webster
tissue: Forebrain
age: E15.5
cell type: Th-EGFP+ sorted dopaminergic neurons

Treatment protocol

Pregnant SW mice were euthanized at E15.5 and the embryos were removed and immediately placed in chilled Eagle’s Minimum Essential Media (EMEM) on ice. Embryos were decapitated and brains were removed into Hank’s Balanced Salt Solution without Mg2+ and Ca2+ (HBSS w/o) on ice. Under a fluorescent microscope, EGFP+ brains were identified and microdissected to yield the desired forebrain (FB) and midbrain (MB) regions desired. Microdissected regions were placed in fresh HBSS w/o on ice, and pooled per litter for dissociation.

Growth protocol

Tg(Th-EGFP)DJ76Gsat mice (Th-EGFP) were generated by the GENSAT Project and purchased through the Mutant Mouse Resource and Research Centers Repository. Colony maintenance matings were between hemizygous male Th-EGFP mice and female Swiss Webster (SW) mice, obtained from Charles River Laboratories. This same mating scheme was used to establish timed matings, generating litters for assay; day on which vaginal plug is observed, E0.5.

Extracted molecule

total RNA

Extraction protocol

Pooled brain regions were dissociated using the Papain Dissociation System (Worthington Biochemical Corporation). The tissue was dissociated in the papain solution for 30 minutes at 37°C, with gentle trituration every 10 minutes using a sterile Pasteur pipette. Following dissociation, cells were passed through a 40µm cell strainer into a 50mL conical, centrifuged for 5 minutes at 300g, resuspended in albumin-inhibitor solution containing DNase, applied to a discontinuous density gradient, and centrifuged for 6 minutes at 70g. The resulting cell pellet was resuspended in HBSS with Mg2+ and Ca2+ and submitted to FACS. Aliquots of 50,000 EGFP+ cells were sorted directly into 300µL HBSS with Mg2+ and Ca2+ with 10% FBS for ATAC-seq. Aliquots containing ≥50,000 EGFP+ cells were sorted into kit-provided lysis buffer for RNA-seq.
Total RNA was extracted using the Purelink RNA Micro Kit (Invitrogen). Following FACS isolation into kit-provided lysis buffer, samples were homogenized and RNA extraction proceeded using manufacturer’s recommendations. Total RNA integrity was determined using the RNA Pico Kit (Agilent). RNA samples were sent to the Sidney Kimmel Comprehensive Cancer Center Next Generation Sequencing Core at Johns Hopkins for library preparation, using the Ovation RNA-Seq System V2 (Nugen), and sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

gencode.vM9_rpkm.txt

Data processing

Libraries were pooled and sequenced on Illumina’s HiSeq 2500 in Rapid Run mode with 2x100bp reads to an average depth of >90 million reads per library. Quality of sequencing was evaluated using FastQC. FASTQ files were aligned to mm9 using HISAT2 (v2.0.1-beta) with --dta specified. Aligned reads from individual samples were quantified against a reference transcriptome using the Rsubread package (v1.22.3) function “featureCounts” with the following options: isPairedEnd = TRUE, requireBothEndsMapped = TRUE, isGTFAnnotationFile = TRUE, useMetaFeature = TRUE. The GENCODE vM9 GTF was downloaded (date: March 30, 2016) and lifted over from the mm10 to the mm9 genome using CrossMap (v0.2.2) with default parameters. This was used for quantification, in which gene-level raw counts were converted to RPKM values and means for each region were calculated.
Supplementary_files_format_and_content: tab-delimited text file containing a matrix of FPKM expression estimates for each gene in each sample
Genome_build: mm9

Submission date

Nov 13, 2018

Last update date

Nov 14, 2018

Contact name

Andy McCallion

E-mail(s)

andy@jhmi.edu

Organization name

Johns Hopkins School of Medicine

Department

Institute of Genetic Medicine