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| Status |
Public on Sep 30, 2023 |
| Title |
atac-seq_heart_male_reference_3_vial_80032885820 |
| Sample type |
SRA |
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| Source name |
ATAC-seq of Rat Male Heart Powder Reference
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| Organism |
Rattus norvegicus |
| Characteristics |
tissue: Heart Powder
treatment: Reference
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed at Stanford University and the Icahn School of Medicine at Mount Sinai. Only Stanford ATAC-seq data were used in this manuscript. Nuclei from aliquoted tissue samples (30 mg for white adipose, 15 mg for brown adipose, 10 mg for hippocampus, kidney, lung, gastrocnemius, heart, and liver tissues) were extracted using the Omni-ATAC protocol with modifications. Two tissue-specific consortium reference standards were included for sample processing QC. The white adipose, brown adipose, and hippocampus tissues were processed using no-douncing nuclei extraction to prevent fat droplets from interfering with the subsequent transposition steps. Tissue powder was incubated in the homogenization buffer for 10 min at 4C. The tube was inverted every 2-3 minutes. The heart, liver, kidney, lung, and gastrocnemius tissues were processed using the Omni-ATAC protocol with modifications. Tissue powder was incubated in the homogenization buffer for 5 minutes on ice and dounced 10 times using pestle A and 20 times with pestle B. For both protocols, the homogenate passed through a 40 µm cell strainer to collect nuclei. Nuclei were stained with DAPI and counted using an automated cell counter. 50,000 nuclei (or max. 500 µl nuclei) were added to 1 ml ATAC-RSB buffer and spun at 1000 g for 10 minutes, and the supernatant was removed. The nuclei pellet was resuspended in 50 µl of transposition mixture and incubated at 37C for 30 minutes with 1000 rpm shaking. The transposed DNA was purified using Qiagen MinElute Purification kits (Qiagen # 28006).
The DNA product was amplified using NEBnext High-Fidelity 2x PCR Master Mix (NEB, M0541L) and custom indexed primers. 1.8x SPRIselect beads were used to clean the PCR reaction and obtain the ATAC-seq library for sequencing.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
motrpac_pass1b-06_t58-heart_epigen-atac-seq_counts.txt.gz
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| Data processing |
Pooled libraries were sequenced on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) to a target depth of 35 million read pairs (70 million paired-end reads) per sample using a paired-end 50 base-pair run configuration
Reads were demultiplexed with bcl2fastq2 (v2.20.0) (Illumina, San Diego, CA, USA)
Data was processed with the ENCODE ATAC-seq pipeline (v1.7.0) (https://github.com/ENCODE-DCC/atac-seq-pipeline)
Samples from a single sex and training time point, e.g., males trained for 2 weeks, were analyzed together as biological replicates in a single workflow
Briefly, adapters were trimmed with cutadapt v2.5 and aligned to release 96 of the Ensembl Rattus norvegicus (rn6) genome with Bowtie 2 v2.3.4.3
Duplicate reads and reads mapping to the mitochondrial chromosome were removed
12 Signal files and peak calls were generated using MACS2 v2.2.4, both from reads from each sample and pooled reads from all biological replicates
Pooled peaks were compared with the peaks called for each replicate individually using Irreproducibility Discovery Rate and thresholded to generate an optimal set of peaks
The cloud implementation of the ENCODE ATAC-seq pipeline and source code for the post-processing steps are available at https://github.com/MoTrPAC/motrpac-atac-seq-pipeline
Optimal peaks (overlap.optimal_peak.narrowPeak.bed.gz) from all workflows were concatenated, trimmed to 200 base pairs around the summit, and sorted and merged with bedtools v2.29.0 to generate a master peak list
This peak list was intersected with the filtered alignments from each sample using bedtools coverage with options -nonamecheck and -counts to generate a peak by sample matrix of raw counts.
Assembly: rn6
Supplementary files format and content: Raw read counts matrix of the number of reads aligning to the genomic region per merged consensus ATAC-seq peak per tissue/phase.
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| Submission date |
Sep 05, 2023 |
| Last update date |
Sep 30, 2023 |
| Contact name |
Euan Ashley |
| Organization name |
Stanford University
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| Street address |
870 Quarry Road
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL25947 |
| Series (2) |
| GSE242357 |
MoTrPAC - Endurance Exercise Training Study In 6 Months Old Rats [ATAC-seq] |
| GSE242358 |
MoTrPAC: Endurance Exercise Training Study In 6 Months Old Rats |
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| Relations |
| BioSample |
SAMN36625363 |
| SRA |
SRX21332455 |
| 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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