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| Status |
Public on Nov 12, 2024 |
| Title |
CTCFdegronPlus_15m_B2_T2 |
| Sample type |
SRA |
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| Source name |
mESCs
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| Organism |
Mus musculus |
| Characteristics |
cell type: mESCs
genotype: CTCF-AID
treatment: EcoGII treated; 15m BrdU; auxin treated
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| Treatment protocol |
Briefly, all nuclei were collected by centrifugation (300xg, 5 min), washed in ice cold 1X PBS, spun again (300xg, 5 min), and resuspended in 1 mL Nuclear Isolation Buffer (20mM HEPES, 10mM KCl, 1mM MgCl2, 0.1% Triton X-100, 20% Glycerol, and 1X Protease Inhibitor (Roche 4693132001)) per 10 million cells by gently pipetting 5x with a wide-bore pipette tip. The suspension was incubated on ice for 5 minutes, and nuclei were pelleted (600xg, 4oC, 5 min), washed with Buffer M (15mM Tris-HCl pH 8.0, 15 mM NaCl, 60mM KCl, 0.5mM Spermidine), and spun once again (600xg, 4oC, 5 min). Nuclei were resuspended in 200 uL of Methylation Reaction Buffer (Buffer M containing 1mM S-adenosylmethionine (SAM; New England BioLabs B9003S)). 10uL high-concentration EcoGII was added per 1e6 nuclei and the nuclei suspension was incubated at 37oC for 30 minutes. An additional 1 uL of 32 mM SAM was supplemented after 15 minutes. Any unmethylated controls were treated similarly with the addition of SAM, but EcoGII was excluded from those reactions.
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| Growth protocol |
K562 cells (ATCC) were grown for at least three days in standard media containing RPMI 1640 + L-Glutamine (Hyclone) supplemented with 10% Fetal Bovine Serum (Gibco) and 1% Penicillin-Streptomycin (Gibco). mESC E14 cells were grown on 0.2% gelatin and maintained in standard media containing DMEM + Glutamax (ThermoFisher 10566-016), 15% Fetal Bovine Serum (ThermoFisher SH30071.03), 1X Non-Essential Amino Acids (ThermoFisher 11140-50), 1 mM Sodium Pyruvate (ThermoFisher 11360-070), 0.128mM 2-Mercaptoethanol (BioRad 1610710XTU), and 1X Leukemia Inhibitory Factor (purified and gifted by Barbara Panning Lab at UCSF). mESCs were passaged every 1-2 days using TrypLE Express Enzyme (1X) (12605010). Media was changed daily when cells were not passaged. All cell lines were regularly tested negative for mycoplasma contamination. Standard media was added to mESC E14 or K562 cells with a final concentration of 10 uM BrdU (abcam ab142567). Cells were incubated in BrdU-containing media for between 5 minutes and 24 hours. After labeling, mESC E14 plates were rinsed three times with 1x PBS and collected using TrypLE Express Enzyme (1X) (12605010). K562s were collected and spun down (300xg, 5 minutes) immediately after labeling.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To the methylation reaction, 2.65 uL 10% SDS and 2.65 uL of 20 mg/mL Proteinase K (Thermo Scientific AM2548) were added and incubated at 65oC for at least 2 hours and up to overnight. To extract the DNA, an equal volume of Phenol-Chloroform was added and mixed vigorously by shaking. The samples were then spun at max speed for 2 min and the aqueous portion was removed. To the aqueous extraction, 0.1x volumes of 3M NaOAc, 1 uL GlycoBlue, and 3x volumes of cold 100% EtOH were added, mixed by inversion and incubated overnight at -20oC or for 2 hours at -80oC. Samples were then spun at max speed for 30 minutes at 4oC, washed with 500 uL 70% EtOH, spun again at max speed for 2 minutes at 4oC. The resulting pellet was air dried and resuspended in 40 uL EB.
Purified DNA from mESCs and K562s was sheared using a Covaris g-tube (520079) in a 5424 rotor at 7,000 RPM for 6 passes for a target size between 6,000 and 8,000 bp. Sheared DNA was used as input for the PacBio HiFi SMRTbell Library Preparation protocol using the SMRTbell Express Template Prep Kit 2.0. Briefly, samples underwent removal of single stranded overhangs, DNA damage repair, end-repair, A-tailing, barcoded SMRTbell adapter ligation, and exonuclease cleanup according to manufacturer’s protocol followed by a 1X AMPure PB bead cleanup. Final sample concentration was measured via Qubit High Sensitivity DNA Assay and library size was measured on an Agilent Bioanalyzer DNA chip. Libraries were sequenced on PacBio Sequel II 8M SMRTcells.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Sequel II |
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| Description |
CTCF-AID mESCs were provided by Elphege Nora's lab (UCSF). Cells were treated with 0.5 mM auxin for 6h prior to any BrdU labeling, after which nuclei were isolated and treated with EcoGII for footprinting of protein-DNA contacts, and DNA was purified and prepared into PacBio SMRTbell libraries for sequencing on the PacBio Sequel II platform.
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| Data processing |
Reads are demultiplexed using lima to produce a .bam file for subreads for each sample.
Circular consensus sequences (CCS) were generated for each sample using ccs to produce a corresponding .bam file per sample.
Supplementary files format and content: Processed data is shared in .pickle format.
Library strategy: Replication-Aware Single-molecule Accessibility Mapping (RASAM)
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| Submission date |
Oct 17, 2023 |
| Last update date |
Nov 12, 2024 |
| Contact name |
Marty G Yang |
| E-mail(s) |
marty.yang@gladstone.ucsf.edu
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| Organization name |
Gladstone Institute for Data Science and Biotechnology
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| Lab |
Vijay Ramani
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| Street address |
1700 Owens Street
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL29177 |
| Series (1) |
| GSE245558 |
The single-molecule accessibility landscape of newly replicated mammalian chromatin |
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| Relations |
| BioSample |
SAMN38001863 |
| SRA |
SRX22242785 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7844894_230419_MO_CTCFdegron_RASAM_15min_treated_rep2_combined_full.pickle.gz |
11.4 Gb |
(ftp)(http) |
PICKLE |
SRA Run Selector |
| Raw data are available in SRA |
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