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| Status |
Public on Oct 06, 2022 |
| Title |
TS38_1_Flag_HMG20B_dmso |
| Sample type |
SRA |
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| Source name |
THP1 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: THP1
chip antibody: Flag (F3165; Sigma)
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| Growth protocol |
Cultured in RPMI 1640 medium (Sigma Aldrich) supplemented with 2mM L-Glutamine (Life Technologies) and 10% fetal bovine serum (Sigma Aldrich).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
10^8 cells were used for each precipitation using the method described by Lee et al. (2006). Briefly, cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature before the reaction was quenched with 0.125M glycine. Cell pellets were washed twice with PBS and nuclear lysates sonicated for 6 cycles using a Bioruptor® Pico (Diagenode, Liege, Belgium). 10μg of antibody bound to 100μl of magnetic beads (Dynabeads Protein G, Invitrogen, Carlsbad, CA) was added to each sample and immunoprecipitation performed overnight on a rotator at 4°C and 20rpm. After five washes with RIPA buffer (50mM HEPES pH 7.6, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5M LiCl), chromatin IP-bound fractions were extracted by incubating for 15 min at 65°C with elution buffer (50mM TrisHCl pH8, 10mM EDTA, 1% SDS). Crosslinking was then reversed by incubation at 65°C for 6 hours. RNaseA (1mg/ml) and proteinase K (20mg/ml) were added to eliminate RNA and protein from the samples. DNA was extracted using phenol:chloroform:isoamyl alcohol and precipitated by adding 2 volumes of ice-cold 100% ethanol, glycogen (20μg/μl), 200mM NaCl and freezing at -80°C for at least 1hr. Pellets were washed with 70% ethanol and eluted in 50μl 10mM TrisHCl pH8.0.
Libraries were prepared for sequencing using a Microplex Library Preparation Kit (Diagenode). 200-800bp fragments were selected using AMPure beads (Beckman Coulter, Brea, CA) and quantified by qPCR with a KAPA Library Quantification Kit (Kapa Biosystems, Basel, Switzerland). Sequencing was performed using a NextSeq desktop sequencing system (Illumina) with 75bp, paired-end high output generating 65-80M reads per sample.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Cultured in RPMI 1640 medium (Sigma Aldrich) supplemented with 2mM L-Glutamine (Life Technologies) and 10% fetal bovine serum (Sigma Aldrich).
Human THP1 AML cells were infected with lentivirus to induce doxycycline-induced expression of Flag-tagged full HMG20B, or a fusion protein containing the GFI1 DNA binding domain (GFI1-ZNF) fused with the full HMG20B. Cells were grown in the presence or absence of doxycycline for 48 hr. and subsequently ChIP-sequencing for LSD1, GFI1, Flag-HMG20B/GFI1-ZNF-HMG20B or H3K27ac was done.
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| Data processing |
Reads were aligned to the human genome (hg38) using BWA-MEM v0.7.15 (5). Reads were further filtered using Bedtools v2.25.0 to keep only reads that mapped to standard chromosomes and to remove reads mapped to blacklisted regions defined by ENCODE (http://mitra.stanford.edu/kundaje).
Genome_build: GRCh38
Supplementary_files_format_and_content: bigwig
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| Submission date |
Jan 03, 2022 |
| Last update date |
Oct 06, 2022 |
| Contact name |
Tim C Somervaille |
| E-mail(s) |
tim.somervaille@cruk.manchester.ac.uk
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| Organization name |
Cancer Research UK
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| Department |
Manchester Institute
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| Lab |
Leukaemia Biology
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| Street address |
Wilmslow Rd
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| City |
Manchester |
| ZIP/Postal code |
M20 4BX |
| Country |
United Kingdom |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE192972 |
HMG20B stabilizes interaction of LSD1 with GFI1 on chromatin to confer transcription repression and leukemia cell differentiation block [ChIP-seq I] |
| GSE192975 |
HMG20B stabilizes interaction of LSD1 with GFI1 on chromatin to confer transcription repression and leukemia cell differentiation block |
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| Relations |
| BioSample |
SAMN24593051 |
| SRA |
SRX13589579 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5770028_TS38_1_Flag_HMG20B_dmso.bw |
696.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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