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| Status |
Public on May 01, 2022 |
| Title |
MNase-seq_NC_rep1 |
| Sample type |
SRA |
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| Source name |
human haploid eHAP cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: eHAP
genotype: wild-type
knockout gene: none
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| Treatment protocol |
Cas9-expressing eHAP cells were transfected with a plasmid expressing sgRNAs against indicated gene and puromycin-resistance gene using FuGENE HD (Promega), and selected with puromycin from 24 hrs post-transfection to 72 hrs. On 5 days post-transfection, cells were used for MNase-seq assays.
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| Growth protocol |
eHAP cells (Horizon Discovery) (Essletzbichler et al., 2014) grown in Iscove’s Modified Dulbecco’s Medium (IMDM, Wako) supplemented with 10% FBS (Sigma) and penicillin/streptomycin (Wako).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To analyze nucleosome occupancy quantitatively in control and TFDP1 knockout cells, We included Drosophila S2 cells as a spike-in control. After mixing 1x106 eHAP cells and 5x104 S2 cells, cells were washed with PBS containing 0.1% BSA and subsequently permeabilized with 0.5% IGEPAL CA-630 in RSB buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 1 mM CaCl2, 3 mM MgCl2 and 0.5 mM PMSF) for 20 min on ice. Nuclei were digested in RSB containing 2.5 U MNase (Takara) at 37 ˚C for 10 min. The reaction was terminated by adding EDTA, EGTA and Proteinase K. The extracted DNA was fractionated by agarose gel electrophoresis, and DNA fragments between 125 and 175 bp representing mono-nucleosome was purified with Monarch PCR & DNA Cleanup Kit followed by treatment with RNase.
Libraries for sequencing were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and sequenced on Illumina HiSeq3000 for 100 bp paired-end reads. Two biological replicates were analyzed for each experimental condition.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
The sequenced reads were quality controlled by Trimmomatic with the option “ILLUMINACLIP:adapter_file:2:30:10 LEADING:20 TRAILING:20 MINLEN:36”.
Bowtie2 with the parameter “-- very-sensitive --maxins 400” mapped MNase-seq reads to the hybrid reference genome (hg38 and dm6).
The reads mapped to multiple positions, chrY, chrM, and blacklisted regions were removed by Samtools (http://www.htslib.org/), and the duplicated reads were filtered by Picard MarkDuplicates.
The scaling factors were calculated by using total mapped read counts for hg38 and dm3, and were used for spike-in normalization.
Genome_build: hg38
Supplementary_files_format_and_content: The read coverage on hg38 were normalized with the scaling factors using bamCoverage with parameters “--binSize 1 --MNase --sclaeFactor”
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| Submission date |
Jan 29, 2020 |
| Last update date |
May 01, 2022 |
| Contact name |
Yusuke Miyanari |
| E-mail(s) |
[email protected]
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| Phone |
81 76 234 4571
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| Organization name |
Kanazawa University
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| Department |
NanoLSI
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| Lab |
Nuclear Dynamics
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| Street address |
Kakuma
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| City |
Kanazawa |
| ZIP/Postal code |
9201192 |
| Country |
Japan |
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| Platform ID |
GPL21290 |
| Series (2) |
| GSE144452 |
MNase-seq analyses with TFDP1 knockout eHAP cells |
| GSE144454 |
A genome wide screening for effectors of chromatin accessibility |
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| Relations |
| BioSample |
SAMN13940261 |
| SRA |
SRX7646671 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4288491_mnase.3.chrM.q20.PCR.bk.hg38.s0.934.NC.bw |
436.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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