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| Status |
Public on Jul 19, 2022 |
| Title |
629_Hi-C |
| Sample type |
SRA |
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| Source name |
Primary AML
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Selected PBMC or BM cells
assay: Hi-C
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| Treatment protocol |
5-azacytidine (MilliporeSigma A2385-100MG) was dissolved in DMSO to make 100mM stock solution, aliquoted and stored in -80°C. Working solutions (0.5uM-10uM) was made from further dilution of stock solution using complete cell culture media. Media was changed every 24 hours with freshly made 5-AZA solution. Dead cells are removed by Ficoll-Paque PLUS density gradient media at the end of cell culture before cells are further processed for any profiling experiments.
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| Growth protocol |
HL60 (ATCC CCL-240) and Kasumi-1 (ATCC CRL-2724) cells were given to us as a gift from Dr Sinisa Dovat lab, which are new vials purchased from ATCC. Cells are cultured following the manufacturer’s culture method. Kasumi is cultured with RPMI-1640 (Gibco, 11875093) with 20% FBS. HL60 is cultured with Iscove's Modified Dulbecco's Medium (IMDM) (ATCC® 30-2005).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
One to two million cryopreserved primary samples or cell lines in cell culture were spinned down with 500g and resuspended in 1ml/million RPMI 1640 medium with 10% fetal bovine serum, immediately crosslinked with 37% formaldehyde (MilliporeSigma 252549) to a final concentration of 2%, and incubated at room temperature for 10 minutes on a tube revolver at 16 rpm to mix, quenched by 2.5M glycine solution with a final concentration of 0.2M and incubated in room temperature for 5 minutes on revolver. Cells were pelleted by centrifuge at 500g at 4°C for five minutes and washed once with 1ml cold 1X PBS by centrifuge at 500g at 4°C for five minutes and the supernatant was discarded. Cells were lysed to extract nuclei with 250ul lysis buffer (10mM Tris-HCl pH8.0, 10mM NaCl, 0.2% Igepal CA630) mixed with 50ul 50x protease inhibitor (Sigma, P8340) and incubated on ice for 15 minutes, centrifuge to pellet at 2500g, 4°C for five minutes and washed with 500ul lysis buffer. Cell pellets were resuspended in 50ul 0.5% sodium dodecyl sulfate (SDS) and incubated at 62°C for 10 minutes, quenched by 145ul water and 25ul 10% Triton X-100 (Sigma, 93443), and incubated at 37°C for 15 minutes. 25μl of 10X NEBuffer2 (NEB B7207) and 100 unit of MboI restriction enzyme (NEB, R0147) was then added to the reaction for overnight DNA digestion at 37°C on the tube revolver. The digestion was then quenched by incubation at 62°C for 20 minutes. DNA was then end repaired and Biotin labeled with 50ul fill-in master mix (37.5μl of 0.4mM biotin-14-dATP (Life Technologies, 19524-016), 1.5μl of 10mM dCTP, 1.5μl of 10mM dGTP, 1.5μl of 10mM dTTP, 8μl of 5U/μl DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210) and incubated at 37°C for 1.5 hours.Then DNA was ligated with 900ul of ligration master mix (669ul water, 120μl of 10X NEB T4 DNA ligase buffer (NEB, B0202), 100μl of 10% Triton X-100, 6μl of 20mg/ml Bovine Serum Albumin (MilliporeSigma B8667), 5μl of 400 U/ μl T4 DNA Ligase (NEB, M0202) ), incubated at room temperature for 4 hours with slow rotation. Decrosslink DNA by adding 50ul of 20mg/ml proteinase K (QIAGEN 19133) and 120ul of 10% SDS, incubated at 55°C for 30 minutes. Quench the reaction by adding 130μl of 5M sodium chloride and incubate at 68°C overnight. Precipitate DNA by adding 1.6X volume of pure ethanol and 0.1X volume of 3M sodium acetate, pH 5.2 (MilliporeSigma S7899), incubate at -80°C for at least half hour, and centrifuge at max speed, 2°C for 15 minutes to discard supernatant. Wash DNA once with 700ul 80% ethanol. Dissolve dried DNA pellet in 130ul 10 mM Tris-HCl, pH 8. Sonicate the solution to shear DNA to average size of 300-500bp with Covaris sonicator, with parameters set as followings: PIP 140, duty factor 10, burst 200, and duration 58s-80s. Run 4ul sheared DNA in 16ul water on a 2% agarose to verify the size.
Pull down biotin-labeled DNA by washing 150μl of 10mg/ml Dynabeads MyOne Streptavidin T1 beads (Life technologies, 65602) with 400μl of 1X Tween Washing Buffer (TWB: 5mM Tris-HCl (pH 7.5); 0.5mM EDTA; 1M NaCl; 0.05% Tween 20), discard the solution. Resuspend the beads in 300μl of 2X Binding Buffer (10mM Tris-HCl (pH 7.5); 1mM EDTA; 2M NaCl) and add to the sheared DNA. Incubate at room temperature for 15 minutes with rotation. Separate beads and discard the supernatant with a magnetic rack. Wash beads with 600ul TWB buffer twice. End repair of sheared DNA by resuspending beads in 100ul 1X NEB T4 DNA ligase buffer (NEB, B0202), separating the beads, and resuspending in end repair master mix (88μl of 1X NEB T4 DNA ligase buffer with 10mM ATP (NEB B0202S), 2μl of 25mM dNTP mix, 5μl of 10U/μl NEB T4 PNK (NEB, M0201), 4μl of 3U/μl NEB T4 DNA polymerase I (NEB, M0203) , 1μl of 5U/μl NEB DNA polymerase I, Large (Klenow) Fragment (NEB, M0210) ), and incubation at room temperature for half hour. Beads were washed twice with 500ul TWB buffer and resuspended in 100μl 1X Quick ligation reaction buffer (NEB, B6058), recollected, and proceeded with dATP attachment by resuspended in 100μl master mix (90μl of 1X NEBuffer 2, 5μl of 10mM dATP, 5μl of 5U/μl NEB Klenow exo minus (NEB, M0212)), incubated at 37°C for 30 minutes. Beads were washed twice with 500ul TWB buffer and resuspended in 100μl 1X Quick ligation reaction buffer (NEB, B6058), recollected, proceeded with adaptor ligation through resuspension in 50μl of 1X NEB Quick ligation reaction buffer, 2μl of NEB DNA Quick ligase (NEB, M2200), and 3ul of Illumina adaptor of choice, incubated in room temperature for 15 minutes. Beads were washed by 600ul TWB buffer and 100ul 1X Tris buffer, resuspended in 50ul 1X Tris buffer, heated on 98°C for 10 minutes to elute the DNA off the beads. Beads were discarded. Size selection was performed to remove small DNA fragments by adding 0.8X-0.9X KAPA beads to the DNA elution, incubation at room temperature for 5 minutes, and beads were collected with supernatant discarded. Wash beads twice with 500ul 80% ethanol and elute beads in 50ul 1X Tris buffer. Library amplification was performed with 4-12 cycles of PCR with KAPA 2X library mix. Size selection was performed to remove small and large fragments using KAPA beads and maintain DNA fragments of 150bp-500bp. Libraries were sequenced as 150 bp paired-end reads with a raw sequencing depth between 300 million to 700 million read pairs per sample on Novaseq.
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| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Library strategy: Hi-C
Paired-end reads were first trimmed by Trim_Galore! (version 0.6.0) to remove adapters and low-quality bases with parameter “--paired”
Trimmed reads were then mapped to human genome GRCh38 by BWA MEM (version 0.7.17-r1198) with parameter “-SP5M”
Mapped reads were deduplicated with “pairtools dedup” (version 0.3.0)
Reads mapped to the same MboI restriction fragment or in outward direction are removed for downstream analysis.
Final pairs files were generated using Pairtools (version 0.3.0)
Genome_build: hg38
Supplementary_files_format_and_content: Pairs format, chromatin interaction intensity for pairs of genomic loci
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| Submission date |
Jun 09, 2020 |
| Last update date |
Jul 19, 2022 |
| Contact name |
Feng Yue |
| Organization name |
Northwestern University
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| Department |
Biochemistry and Molecular Genetics
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| Street address |
303 E Chicago Ave. Simpson Querrey 7-518
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE152135 |
Subtype-Specific and Structure Variation-Induced Chromatin Spatial Reorganization in Acute Myeloid Leukemia [Hi-C] |
| GSE152136 |
Subtype-Specific and Structure Variation-Induced Chromatin Spatial Reorganization in Acute Myeloid Leukemia |
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| Relations |
| BioSample |
SAMN15192059 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4604274_629.iced.mcool |
819.6 Mb |
(ftp)(http) |
MCOOL |
| GSM4604274_629_Hi-C.nodups.pairs.gz |
5.4 Gb |
(ftp)(http) |
PAIRS |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
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