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| Status |
Public on Apr 25, 2020 |
| Title |
atac-18gw_cge |
| Sample type |
SRA |
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| Source name |
Caudal ganglionic eminence
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| Organism |
Homo sapiens |
| Characteristics |
gestational weeks: 18
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| Treatment protocol |
none
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| Growth protocol |
none, fresh intact samples from human specimens were used
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ATAC-seq samples, from each dissection intact nuclei were isolated by manually douncing the tissue twenty times in 1mL Buffer 1 (300mM sucrose, 60mM KCl, 15mM NaCl, 15mM Tris-HCl, pH 7.5, 5mM MgCl2, 0.1mM EGTA, 1mM DTT, 1.1mM PMSF, Protease inhibitors) on ice using a loose pestle douncer, and then lysed on ice for 10 minutes after adding 1mL Buffer 2 (300mM sucrose, 60mM KCl, 15mM NaCl, 15mM Tris-HCl, pH 7.5, 5mM MgCl2, 0.1mM EGTA, 0.1% NP-40, 1mM DTT, 1.1mM PMSF, Protease inhibitors). During this ten minutes, nuclei were counted using trypan blue and 50,000 nuclei were spun down at 7,000rpm for ten minutes at 4C. Nuclei were resuspended in 25uL Tagmentation buffer, 22.5 uL Nuclease Free H20, and 2.5 uL Tagmentation Enzyme from Illumina DNA Kit (number), gently mixed, and placed in 37C water bath for thirty minutes. The tagmentation reaction was stopped by MinElute PCR purification and DNA was eluted in 10uL Nuclease Free water. For ChIP-seq samples, intact nuclei were isolated by manually douncing the tissue twenty times in 1mL Buffer 1 (300mM sucrose, 60mM KCl, 15mM NaCl, 15mM Tris-HCl, pH 7.5, 5mM MgCl2, 0.1mM EGTA, 1mM DTT, 1.1mM PMSF, 50mM Sodium Butyrate, EDTA-free Protease inhibitors) on ice using a loose pestle douncer, and then lysed on ice for 10 minutes after adding 1mL Buffer 2 (300mM sucrose, 60mM KCl, 15mM NaCl, 15mM Tris-HCl, pH 7.5, 5mM MgCl2, 0.1mM EGTA, 0.1% NP-40, 1mM DTT, 1.1mM PMSF, 50mM Sodium Butyrate, EDTA-free Protease inhibitors). During this ten minutes, nuclei were counted using trypan blue and 500,000 nuclei were spun down at 7,000rpm for ten minutes at 4C. Nuclei were resuspended in 0.250mL MNase buffer (320mM sucrose, 50mM Tris-HCl, pH 7.5, 4mM MgCl2, 1mM CaCl2, 1.1mM PMSF, 50mM Sodium Butyrate) and incubated in a 37C water bath with 2 microliters MNase enzyme (NEB) for eight minutes. MNase digestion was stopped by adding 10 microliters 0.5M EDTA, and chromatin was spun down for 10 minutes 10,000rpm 4C. Soluble fraction S1 supernatant was saved at 4C overnight, and S2 fraction was dialyzed overnight in 250uL dialysis buffer at 4C (1mm Tris-HCl pH 7.5, 0.2mM EDTA, 0.1mM PMSF, 50mM Sodium Butyrate, Protease Inhibitors). S1 and S2 fractions were combined, 50 microliters were saved as input, and Chromatin immunoprecipitation was set up in 50mM Tris, pH 7.5, 10mM EDTA, 125 mM NaCl1, 0.1% Tween. 250m M Sodium Butyrate was supplemented for H3K27ac ChIPs. The following antibodies were used for ChIP: H3K27ac (Millipore, cma309), H3K4me1 (Abcam, ab8895), H3K27me3 (Millipore, 07-449), H4K20me3 (Abcam, ab9053), and H3K9me3 (Abcan, ab8898). 1 microliter of antibody was added to 1mL chromatin in ChIP buffer and incubated overnight with chromatin at 4C rotating. Protein A and Protein G beads (10 microliters each) were blocked overnight in 700uL ChIP buffer, 20 uL yeast tRNA (20mg/mL), and 300uL BSA (10mg/mL). Beads were washed three times on ice in Wash buffer 1 (50 mM Tris, pH 7.5 , 10mM EDTA , 125mM NaCl, 0.1% Tween-20, with protease inhibitors and 5mM sodium butyrate) and three times in Wash buffer 2 ((50 mM Tris, pH 7.5 , 10mM EDTA , 175mM NaCl, 0.1% NP-40, with protease inhibitors and 5mM sodium butyrate), and ChIP DNA was eluted in elution buffer at 37C and purified by phenol chloroform extraction and ethanol precipitation.
ATAC-seq library generation was performed using Illumina barcode oligos as described (Buenrostro et al 2015), for 8-11 cycles PCR. The number of cycles was empirically determined for each library. ChIP-seq sequencing lIbraries were made using Nugen Ovation Ultralow kit and quantified by bioanalyzer.
Libraries were bioanalyzed using Agilent High Sensitivity DNA Kit, pooled together and sequenced on Hiseq 2500 using paired end sequencing.
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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 |
Data was processed using the ENCODE ATAC-seq and ChIP-seq pipelines.
Predicted regulatory elements were generated using custom Python code.
Genome_build: hg19, hg38
Supplementary_files_format_and_content: ATAC-seq and ChIP-seq signal (fold change over control) in bigWig format, ATAC-seq and ChIP-seq peaks (optimal IDR threshold) in narrowPeak format, OCR and pRE annotations in bed format
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| Submission date |
Apr 24, 2020 |
| Last update date |
Apr 25, 2020 |
| Contact name |
John Rubenstein |
| Organization name |
University of California, San Francisco
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| Department |
Department of Psychiatry
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| Street address |
401 Parnassus Ave
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143 |
| Country |
USA |
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| Platform ID |
GPL27467 |
| Series (1) |
| GSE149268 |
A Chromatin Accessibility Atlas of the Developing Human Telencephalon |
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| Relations |
| BioSample |
SAMN14687981 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4495213_atac-18gw_cge-optimal_idr-hg19.narrowPeak.gz |
1.1 Mb |
(ftp)(http) |
NARROWPEAK |
| GSM4495213_atac-18gw_cge-optimal_idr-hg38.narrowPeak.gz |
1.1 Mb |
(ftp)(http) |
NARROWPEAK |
| GSM4495213_atac-18gw_cge-rep1-nodup-fold_change-hg19.bigwig |
125.7 Mb |
(ftp)(http) |
BIGWIG |
| GSM4495213_atac-18gw_cge-rep1-nodup-fold_change-hg38.bigwig |
99.2 Mb |
(ftp)(http) |
BIGWIG |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
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