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Sample GSM3743874 Query DataSets for GSM3743874
Status Public on Jun 08, 2020
Title PSC_WT_Rep1_ATAC-seq
Sample type SRA
 
Source name Pluripotent stem cells
Organism Macaca mulatta
Characteristics cell type: Pluripotent stem cells
disease state: wild type
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
 
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2500
 
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.
 
Submission date May 01, 2019
Last update date Jun 10, 2020
Contact name Isaac Kremsky
E-mail(s) ikremsk@emory.edu
Organization name Emory University
Department Biology
Lab Corces
Street address 1510 Clifton Rd
City Atlanta
State/province GA
ZIP/Postal code 30322
Country USA
 
Platform ID GPL19129
Series (1)
GSE130570 Chromatin Accessibility and Transcription Dynamics During In Vitro Astrocyte Differentiation of Huntington’s Disease Monkey Pluripotent Stem Cells.
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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