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| Status |
Public on Jun 01, 2020 |
| Title |
CA1a_Inp_L_r2_R1 |
| Sample type |
SRA |
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| Source name |
Human breast epithelial cells, virally transformed
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Human breast epithelial cells
cell type: MCF10A-CA1A
treatment: none
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| Treatment protocol |
For EMT induction, MCF-10A cells were treated with 5 ng/ml TGF-β1 (Redsystems) for 8 days. MCF-10A cells were transduced with the lentviral vector pLVTHM shH2A.Z as previously described (Domaschenz, 2017). GFP-positive cells were sorted 2 days post-transduction and further cultured for 8 days before being processed.
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| Growth protocol |
MCF-10A and MCF-10Ca1a cell lines were grown in DMEM/Nutrient F12 (DMEM/F12) media supplemented with 5% Horse Serum, 14 mM NaHCO3, 10 µg/mL insulin, 2 mM L-glutamine, 20 ng/mL human EGF, 500 ng/mL Hydrocortisone and 100 ng/mL Cholera Toxin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei were isolated in nucleus isolation buffer containing: 10 mM HEPES at pH 7.8, 2 mM MgOAc2, 0.3 M sucrose, 1 mM CaCl2, and 1% Nonidet P-40. The nuclei were then pelleted by centrifugation at 1000g for 5 min at 4°C.
Using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (NEB #E7370S/L), DNA sequencing libraries were prepared for the mononucleosomal-sized and sub-nucleosomal-sized fragments for each sample. DNA was end-prepped using NEB Prep enzyme mix, end-repair reaction buffer (10X), and 30 ng of DNA for each samples, then held at 30 degrees Celsius for 30 minutes and then at 65 degrees Celsius for 30 minutes. Adaptors were ligated onto the end-repaired samples by adding NEB Blunt/TA Ligase Master Mix, NEBNext Adaptor for Illumina, and Ligation Enhancer and incubating at 20 degrees Celsius for 15 minutes. The adaptor-ligated DNA was cleaned up using AMPure XP beads to remove any unwanted ligated products. The universal and indexed sequences were added by PCR using 23 ul of adaptor-ligated DNA fragments, NEBNext High Fidelity 2X PCR Master Mix, index primers provided in NEBNext Multiplex (NEB #E7335, #E7500) Oligos for Illumina, and Universal PCR Primers provided in NEBNext Multiplex (NEB #E7335, #E7500) Oligos for Illumina. Then PCR was done for 8 cycles (not including the initial denaturation and final extension). The adaptor-ligated DNA was cleaned up using AMPure XP beads to remove any unwanted products. The libraries were quality-checked using Agilent 2100 Bioanalyzer High-Sensitivity. Across the libraries, the samples ranged between 200-400bp and there were no adapter or primer dimers.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Light MNase digest
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| Data processing |
Basecalls performed with CAVASA version v1.8.4
Sequencing data aligned to the HG19 genome using bowtie2 version 2.2.2, --wrapper basic -0 --end-to-end -p 23 --no-mixed --no-discordant -I 0 -X 500 -q -x -S
Using samtools via the R package gyro (https://github.com/dvera/gyro), raw files converted from sam to bam files, minQual=10
Genome_build: hg19
Supplementary_files_format_and_content: Bed files were generated using bedtools via the R package travis (https://github.com/dvera/travis) and mat10 files were created using the package genmat (https://github.com/dvera/genmat). Nucleosome distributions were calculated by fragments per million for each bp in the 2kb surrounding each TSS.
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| Submission date |
Jul 15, 2019 |
| Last update date |
Nov 07, 2023 |
| Contact name |
LAUREN A COLE |
| E-mail(s) |
LC13G@MY.FSU.EDU
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| Phone |
8506458573
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| Organization name |
FLORIDA STATE UNIVERSITY
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| Street address |
319 STADIUM DR
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| City |
TALLAHASSEE |
| State/province |
Florida |
| ZIP/Postal code |
32304 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE134297 |
Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells (Mnase-seq & ChIP-seq) |
| GSE134299 |
Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells |
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| Relations |
| Reanalyzed by |
GSE247171 |
| BioSample |
SAMN12276479 |
| SRA |
SRX6445042 |
| 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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