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| Status |
Public on Feb 07, 2022 |
| Title |
Skeletal Muscle_Rat2 |
| Sample type |
SRA |
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| Source name |
Gastrocnemius muscle
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: Fischer 344
age: 8 months
Sex: male
tissue: Gastrocnemius muscle
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| Treatment protocol |
For tissue collection, rats were anesthetized via isoflurane, secondary euthanasia was performed via decapitation, and gastrocnemius muscle (skeletal muscle) and abdominal white adipose tissue (adipose tissue) were dissected out and quickly frozen in liquid nitrogen and stored at -80C until nuclei extraction.
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| Growth protocol |
All animal procedures in this study were conducted in accordance with the guidelines of University of Florida for the care and use of laboratory animals (IACUC # 201709757). Rats were obtained from the National Institute on Aging colony at 8 months of age. They were allowed 2 weeks to acclimate to a reverse light cycle (lights off at 8am: lights on at 8pm).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tissue samples were transferred to 1.1 ml micronic tubes (Micronic, USA) and resuspended in 600 µl of homogenization buffer (320 mM Sucrose, 10 mM Tris-HCl pH 7.8, 100 nM EDTA, 5 mM CaCl2, 3 mM Mg(AC)2, 167 µM β-mercaptoethanol, 1X protease inhibitor). Three, 2.8 mm ceramic beads (Omni International, Kennesaw, GA, USA) were added to each micronic tube. Tubes were vortexed briefly and kept on ice. The tissue samples were homogenized in a Bead Ruptor Elite bead mill homogenizer with Omni BR Cryo cooling unit (Omni International) using 2 cycles of the following settings: speed: 1.0, time: 20 sec and speed: 2.1, time: 20 sec, dwell time: 20 sec at both speeds, while cooled by liquid nitrogen to 10°C. Following homogenization, each tissue homogenate was filtered through a mini 20 µm pluriStrainer (pluriSelect, Leipzig, Germany). The number of nuclei in the filtrate was counted using an Automated Cell Counter (Cellometer K2 Image cytometer, Nexcelom) following staining with the Propidium Iodide (PI) dye.
Approximately 75,000 nuclei were transferred into a fresh tube and diluted in 1 ml of ATAC-seq resuspension buffer [ATAC-seq RSB (Corces et al., 2017) + 0.1% Tween 20 (Sigma, St. Louis, MO, USA)]. Nuclei were centrifuged using Centrifuge 5418 R (Eppendorf) at 725 RCF for 10 minutes, and the supernatant was carefully aspirated. Nuclei were incubated in 50 µl transposition reaction mixture containing 25 µl of 2X TD buffer (Illumina, Inc., San Diego, CA, United States), 16.6 µl of PBS, 0.5 µl of 1% digitonin (Sigma), 0.5 µl of 10% Tween-20, 1.5 µl of Tn5 transposase (Illumina, 0.8 U/μl), and 6 µl of nuclease-free water using a thermomixer (USA Scientific, Ocala, FL, USA) set at 37 °C for 30 min with a speed of 1000 rpm. Transposed DNA was purified using a DNA clean and concentrator kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s protocol. Following PCR amplification, libraries were purified using Agencourt Ampure XP beads (Beckman Coulter, Indianapolis, IN, USA) double-size selection (0.5x:1.3x). Library quality was assessed using the Agilent Bioanalyzer High-Sensitivity DNA kit and the Qubit dsDNA high sensitivity assay kit (Thermo Fisher). All libraries were pooled and sequenced with 100-bp paired-end reads on the Illumina Novaseq 6000 at the New York Genome Center.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The ATAC-seq data were processed (trimmed, aligned, filtered, and quality controlled) using the Encode ATAC-seq pipeline v1.7.0 (https://www.encodeproject.org/atac-seq/).
For all samples, read quality was assessed using FastQC (Andrews, 2021).
Trimmomatic (Bolger et al., 2014) was used to remove adapters and low-quality base pairs and reads identified by FastQC.
Reads for each sample were aligned to the rat genome (rn6.0) using Bowtie2 (Li and Durbin, 2010).
After alignment, we removed reads mapping to the mitochondrial genome. As a general measure of sensitivity to Tn5 transposase fragmentation, ATAC-seq signal was defined as a number of transposase cuts mapping to each bp (or a total count of cuts mapping to a selected genomic interval). The cuts were defined as 5’ ends of ATAC-seq reads, with additional shifting by +4 bp and -5bp for reads mapping to the plus and minus strands, respectively (Buenrostro et al., 2013).
Duplications were removed by Picard (https://broadinstitute.github.io/picard/). MACS2 (Zhang et al., 2008) was applied on each merged bam file to call peaks (with option --nomodel --extsize 200 --shift 100).
Genome_build: rn6
Supplementary_files_format_and_content: Peak files
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| Submission date |
Apr 12, 2021 |
| Last update date |
Feb 07, 2022 |
| Contact name |
Venugopalan Nair |
| E-mail(s) |
venugopalan.nair@mssm.edu
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| Phone |
212-241-5809
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| Organization name |
Icahn School of Medicine at Mount Sinai
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| Department |
Neurology
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| Street address |
1468 Madison Avenue
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL25947 |
| Series (1) |
| GSE171923 |
Differential analysis of chromatin accessibility and gene expression profiles identifies cis-regulatory elements in rat adipose and muscle |
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| Relations |
| BioSample |
SAMN18716187 |
| SRA |
SRX10580930 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5237463_RM2_ATAC.narrowPeak.gz |
4.7 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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