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Sample GSM3466723 Query DataSets for GSM3466723
Status Public on Nov 14, 2018
Title MB_RNA_rep2
Sample type SRA
Source name E15.5 midbrain dopaminergic neurons
Organism Mus musculus
Characteristics strain: Tg(Th-EGFP)DJ76Gsat x Swiss Webster
tissue: Midbrain
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
Organization name Johns Hopkins School of Medicine
Department Institute of Genetic Medicine
Street address 733 N Broadway, MRB 446
City Baltimore
State/province MD
ZIP/Postal code 21205
Country USA
Platform ID GPL17021
Series (1)
GSE122450 Parkinson-associated SNCA enhancer variants revealed by open chromatin in mouse dopamine neurons
BioSample SAMN10413960
SRA SRX5001319

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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