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| Status |
Public on Nov 14, 2018 |
| Title |
FB_ATAC_rep3 |
| Sample type |
SRA |
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| Source name |
E15.5 forebrain dopaminergic neurons
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| Organism |
Mus musculus |
| Characteristics |
strain: Tg(Th-EGFP)DJ76Gsat x Swiss Webster
tissue: Forebrain
age: E15.5
cell type: Th-EGFP+ sorted dopaminergic neurons
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| 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.
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| 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.
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| Extracted molecule |
genomic DNA |
| 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.
ATAC-seq library preparation generally follows the steps as set out in the original ATAC-seq paper with minor modifications. Aliquots of 50,000 EGFP+ cells were centrifuged for 5 minutes at 4°C and 500g, washed with 50µL of chilled PBS and centrifuged again for 5 minutes at 4°C and 500g. The cell pellet was resuspended in lysis buffer, as set out in the protocol, and cells were left to lyse for 5 minutes at 4°C before being centrifuged for 10 minutes at 4°C at 500g. The resulting nuclei pellet was transposed, as written, using the transposase from the Nextera DNA Library Preparation Kit. Following transposition, DNA was purified with the MinElute Reaction Clean-up Kit (Qiagen) and eluted in 10µL elution buffer. Libraries were amplified according to the original ATAC-seq protocol. The qPCR surveillance steps were modified such that the additional number of cycles of amplification were calculated as ¼ maximum intensity, so as to limit PCR duplication rates in the final libraries. Amplified libraries were purified with Ampure XP beads (Beckman Coulter) following the Nextera DNA Library Prep Protocol Guide. Libraries were quantified using the Qubit dsDNA High Sensitivity Assay (Invitrogen) in combination with the Agilent 2100 Bioanalyzer using the High Sensitivity DNA Assay (Agilent).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Individual ATAC-seq libraries were sequenced on the Illumina MiSeq to a minimum depth of 20 million, 2x75bp reads per library. A single MB ATAC-seq library was sequenced on the Illumina HiSeq in Rapid Run mode with 2x100bp reads, to a depth of ≥350 million paired-end reads. Quality of sequencing was evaluated using FastQC (v0.11.2; https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were aligned to mm9 using Bowtie2 (v2.2.5), under --local mode. Reads aligning to the mitochondrial genome, unknown and random chromosomes, and PCR duplicates were removed prior to peak calling (SAMtools). Peaks were called on individual libraries and on a concatenated file combining all MB and all FB libraries (“Joint”) using MACS2 (v2.1.1.20160309) “callpeak” with options: --nomodel --nolambda -B -f BAMPE --gsize mm --keep-dup all.
Supplementary_files_format_and_content: tab-delimited text file containing the peak calls from MACS2
Genome_build: mm9
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| Submission date |
Nov 13, 2018 |
| Last update date |
Nov 14, 2018 |
| Contact name |
Andy McCallion |
| E-mail(s) |
andy@jhmi.edu
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| Organization name |
Johns Hopkins School of Medicine
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| Department |
Institute of Genetic Medicine
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| Street address |
733 N Broadway, MRB 446
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
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| Platform ID |
GPL16417 |
| Series (1) |
| GSE122450 |
Parkinson-associated SNCA enhancer variants revealed by open chromatin in mouse dopamine neurons |
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| Relations |
| BioSample |
SAMN10413962 |
| SRA |
SRX5001317 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3466721_FB3_peaks.txt.gz |
1.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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