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| Status |
Public on Jul 28, 2020 |
| Title |
K2C ATAC-Seq |
| Sample type |
SRA |
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| Source name |
Cortical neuron culture
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
age: Embryonic day 18, 11 DIV
treatment: 25mM KCl
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| Treatment protocol |
Neurons were treated as described with neurobasal media (vehicle) or potassium chloride (KCl; 25mM) for 1hr on DIV 11. 50,000 cells per sample were harvested for ATAC-seq.
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| Growth protocol |
Neurons were removed from cortical, hippocampal, or striatal tissue at embryonic day 18, and maintained in vitro for 11 days prior to collection of RNA. Each culture well contained ~250,000 cells.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Following treatment, media containing KCl or Vehicle was aspirated, and cells were washed with 1x cold PBS. Then, 600 µl of Lysis Buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630, Molecular Grade H2O) was added to cell culture wells and incubated for 5 mins on ice. Lysed cells were then transferred to a 1.5 ml Eppendorf tube and centrifuged at 500 g for 10 mins in a swinging bucket centrifuge. Following removal of supernatant containing lysis buffer and cell debris, 500 µl of 1x cold PBS was added to each tube, and nuclei were counted using the Countess II (Life Technologies). The approximate volume needed to achieve 50,000 nuclei was then placed in new Eppendorf tube, and centrifuged at 500 g for 10 mins in a swinging bucket centrifuge.
The nuclei pellet was then resuspended in 22.5 µl of tagmentation reaction mix containing Molecular Grade H2O, 2x Tagment DNA Buffer (Illumina), 0.011% Digitonin, 0.1% Tween-20, followed by the addition of 2.5 µl TDE1 (Illumina) and incubated at 37°C for 1 hr. Following tagmentation, libraries were PCR amplified and purified using the Qiagen Minelute PCR purification kit (Qiagen). To remove primer dimers, libraries were bead purified using one round of 1.0X Ampure XP beads with 80% ethanol, followed by a second round of 0.8X Ampure beads with 80% ethanol. Libraries were sequenced (75-bp paired end reads) on the NextSeq500 platform (Illumina).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ATAC-seq
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| Data processing |
Low-quality bases (Phred <20) and Nextera adapters (5’-CTGTCTCTTATA-3’) were identified and trimmed using FastQC and TrimGalore (v0.4.5).
FASTQ files containing trimmed sequences were then aligned to Rn6 Ensembl genome assembly (v95) to generate binary alignment map (BAM) files with Bowtie2 (v.2.3.4.2) with custom options: `--local --very-sensitive-local`, and `-X 3000`
Post-trimming QC with FastQC
Sequences ordered by genomic position using Samtools (v.1.9)
BAM files for each brain region were merged to generate three metasamples that were used for downstream data analysis. Peaks for each region were called using macs2 (Zhang et al., 2008) callpeak with the options - -qvalue 0.00001 - -gsize 2729862089 - -format BAMPE, ignoring duplicates.
Within each brain region, peaks closer than 1000bps were merged with bedtools and peaks less than 146bp, the specific length of DNA wrapped around a single nucleosome, were removed in R. Finally, peaks from each brain region were merged with bedtools to create an additive peak set containing 192,830 peaks.
Genome_build: rn6
Supplementary_files_format_and_content: BED file cotaining ATAC-seq peaks (output of MACS2). Data are in BED format - columns are chromosome, start, end, and score for how many cell types (of 3) contained the ATAC peak.
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| Submission date |
May 14, 2020 |
| Last update date |
Jul 28, 2020 |
| Contact name |
Jeremy Jason Day |
| E-mail(s) |
jjday@uab.edu
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| Phone |
205-996-8960
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| Organization name |
UAB
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| Department |
Neurobiology
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| Lab |
Day
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| Street address |
1825 University Blvd
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| City |
Birmingham |
| State/province |
AL |
| ZIP/Postal code |
35294 |
| Country |
USA |
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| Platform ID |
GPL20084 |
| Series (1) |
| GSE150589 |
ATAC-seq datasets for chromatin accessibility quantification in "Enhancer RNAs predict enhancer-gene regulatory links and are critical for enhancer function in neuronal systems" |
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| Relations |
| BioSample |
SAMN14925263 |
| SRA |
SRX8342841 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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