|
| Status |
Public on Apr 26, 2022 |
| Title |
4Cseq_293T-EAF1mut_HIST2H2AB-bait_Rep1_R1 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T EAF1-mutant
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
genotype: EAF1-mutant
spike-in source: n/a
protocol/antibody: 4C-seq, Krijger et al Methods, 170: 17-32; doi: 10.1016/j.ymeth.2019.07.014
sequenced molecule: 4C-template DNA
|
| Growth protocol |
Cells were cultured in 1X DMEM (Nacalai tesque) supplemented with 10% fetal bovine serum and 1X penicillin/streptomycin (Nacalai tesque) in a CO2 incubator (5% CO2) at 37°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For 4C-seq, HEK293T cells cultured in 10 cm dish were fixed with 4% formaldehyde for 10 min at room temperature. Cross-linked HEK293T cells were lysed, and the lysate was subjected to first restriction enzyme digestion using NlaIII (R0125S; NEB) followed by in situ ligation reaction using T4 DNA ligase (10799009001; Roche Diagnostics, Tokyo). After reverse-cross-linking, the ligated DNA templates were purified using P-beads (NucleoMag® NGS Clean-up and Size Select, 744970.5, Takara Bio Inc). Then, 2nd round of restriction enzyme digestion using DpnII (R0543S; NEB) and ligation was performed for trimming and circularization of template DNA. Resulting 4C-templates were purified by P-beads and subjected to sequencing library preparation.
4C-seq libraries werec generated essentially as described in Krijger et al., 2020 (Methods, 170: 17-32; doi: 10.1016/j.ymeth.2019.07.014). 4C-seq libralies were pooled and sequenced on Illumina Hiseq 2500 platform.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Library strategy: 4C-seq
Basecalling and bcl to fastq generation was done using bcl2fastq2 v2.18 from illumina.
For 4C-seq, pipe4C was used with the default setting for the data analysis. R1 files of paired-end read files were used for 4C-seq data analysis.
Supplementary_files_format_and_content: BigWig files include normalized count data
|
| |
|
| Submission date |
Jan 02, 2021 |
| Last update date |
Apr 26, 2022 |
| Contact name |
Hidefumi Suzuki |
| E-mail(s) |
h_suzuki@yokohama-cu.ac.jp
|
| Organization name |
Yokohama City University
|
| Street address |
Fukuura 3-9
|
| City |
Yokohama |
| ZIP/Postal code |
236-0004 |
| Country |
Japan |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE164144 |
The 3' Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies |
|
| Relations |
| BioSample |
SAMN17202732 |
| SRA |
SRX9768283 |
