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Status |
Public on Jun 08, 2020 |
Title |
PSC_WT_Rep1_ATAC-seq |
Sample type |
SRA |
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Source name |
Pluripotent stem cells
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Organism |
Macaca mulatta |
Characteristics |
cell type: Pluripotent stem cells disease state: wild type
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Extracted molecule |
genomic DNA |
Extraction protocol |
ell cultures were harvested, washed in cold 1x PBS, and counted. One hundred thousand cells were resuspended in 50 µL cold resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2) containing 0.1% NP-40 (Sigma), 0.1% Tween-20 (Sigma), and 0.01% digitonin (Abcam ab141501) and incubated for 3 min on ice. Following lysis, 1 mL RSB with 0.01% Tween-20 was added to the samples. Samples were centrifuged for 10 min at 4°C/500 x g and the supernatant was carefully removed. Nuclei were then resuspended in the transposase reaction mix (25 µL 2x TD buffer, 2.5 µL Tn5 transposase, 0.5 µL 10% Tween-20, 2.5 µL 1% digitonin [0.05% final concentration], and 19.5 µL water) and incubated at 37°C for 30 min in a thermomixer with shaking at 600 r.p.m. Following the reaction, samples were treated with Proteinase K (Fisher 25530015) at 55°C for 2 hr and genomic DNA was isolated via phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation. Library preparation was performed using 2x KAPA HiFi mix (KAPA BIOSYSTEMS INC #kk4604) and 1 mM indexed primers under the following PCR conditions: 72°C for 5 min; 98°C for 30 sec; and 8-12 cycles at 98°C for 10 sec, 63°C for 30 sec, and 72°C for 1 min. ATAC-seq experiments were performed on two replicates
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Library strategy |
ATAC-seq |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
ATAC-seq raw reads were trimmed using pyadapter_trim.py. Reads were aligned to the “MacaM_Rhesus_Genome_Annotation_v7.8.2” genome using Tophat2 v2.1.0 with the flags, “--no-mixed –no-discordant” for RNA-seq, and using bowtie version 2 2.2.6 with the flag, “-X 2000” for ATAC-seq. Non-uniquely mapped reads were excluded from both RNA-seq and ATAC-seq samples, and duplicates were removed from ATAC-seq samples using picard-tools-2.1.7 MarkDuplicates. For ATAC-seq, only fragments < 115 bp in length were kept, as those indicate transcription factor binding. .bam files were converted to .bed using “bamToBed -split” for RNA-seq, and .bedpe using “bamToBed -bedpe” for ATAC-seq. The ATAC-seq .bedpe file was converted to a bed file containing only the beginning and end of each fragment. .bed files were converted to bedGraph using “bedtools genomecov -scale”, where the scale factor was the total number of uniquely mapped reads for RNA-seq, and the total number of uniquely mapped, non-duplicate fragments for ATAC-seq. The result is an RPM-normalized .bedGraph file for each replicate. .bedGraph files were then converted to bigwig using “bedGraphToBigWig” Genome_build: MacaM_Rhesus_Genome_Annotation_v7.8.2 Supplementary_files_format_and_content: bigwig files of RPM-normalized signal.
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Submission date |
May 01, 2019 |
Last update date |
Jun 10, 2020 |
Contact name |
Isaac Kremsky |
E-mail(s) |
ikremsk@emory.edu
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Organization name |
Emory University
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Department |
Biology
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Lab |
Corces
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Street address |
1510 Clifton Rd
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City |
Atlanta |
State/province |
GA |
ZIP/Postal code |
30322 |
Country |
USA |
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Platform ID |
GPL19129 |
Series (1) |
GSE130570 |
Chromatin Accessibility and Transcription Dynamics During In Vitro Astrocyte Differentiation of Huntington’s Disease Monkey Pluripotent Stem Cells. |
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Relations |
BioSample |
SAMN11552767 |
SRA |
SRX5775622 |
Supplementary file |
Size |
Download |
File type/resource |
GSM3743874_WT_ESC_R1.sorted.duprmvd.TFs.bw |
204.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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