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| Status |
Public on Feb 02, 2024 |
| Title |
tIMEC_H3K4me3 |
| Sample type |
SRA |
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| Source name |
tIMEC
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| Organism |
Homo sapiens |
| Characteristics |
cell line: tIMEC
cell type: mammary epthelial cell-derived cell line
antibody: H3K4me3 (07-473, Millipore)
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| Growth protocol |
For each antibody, 250k cells were collected and mixed with 10k NIH 3T3 cells for the spike-in.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
All cells required for one experiment were spun down for 3 min at 200 x g at RT, supernatant was removed and cell pellet was resuspended in 1.5 ml RT Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, EDTA-free protease inhibitor) by gently pipetting, spun down and washed twice more. After the last wash, supernatant was removed, the pellet was resuspended in 1 ml RT Wash Buffer. ConcavalinA-coated magnetic beads were activated by washing once in binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2 and 1 mM MnCl2) and added to the sample while gently vortexing at RT. Samples were incubated on a rotator for 10 min at RT. Sample was divided in aliquots (250k cells / ab) and placed on a magnet to remove the supernatant. 100 ul of antibody buffer + corresponding antibody (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.025% Digitonin, 4 mM EDTA and EDTA-free protease inhibitor) were pipetted along the sides of the tubes and pipetted up and down 10x. The following antibodies were used at a dilution 1:100: H3K27ac (ab4729, abcam), H3K4me1 (ab8895, abcam), MED1 (ab64965, abcam), H3K4me3 (07-473, Millipore), SOX9 (AB5535, Millipore) and IgG ( . The resuspended beads were rotated o/n at 4°C. The next day, tubes were quickly spun, put on a magnetic rack and supernatant was removed. 150 ul of Dig-wash buffer antibody (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.025% Digitonin, and EDTA-free protease inhibitor) were added and tubes mixed by inversion for four times. Supernatant was removed and 150 ul Protein-A-MNase (700 ng/ml) were added along the tube sites, mixed by pipetting and incubated for 1 h at 4°C on the rotator. Supernatant was removed, samples were washed four times with 150 ul Dig-wash buffer, resuspended in 100 ul Dig-wash buffer and stored in wet ice (0°C) to chill down. 2 ul of 100 mM CaCl2 were added and digestion was incubated for 30 min. Digestion reaction was stopped by adding 100 ul 2x STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 100 ug/ml RNAse A and 50 ug/ml Glycogen). After addition, samples were incubated at 37°C for 30 min for the release of soluble fragments to the supernatant, which was transferred to a new tube. Released DNA was purified by phenol-chloroform extraction.
Sequencing libraries were produced using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645) as described by the manufacturer
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw reads were quality controlled using FastQC and trimmed using Trimmomatic v0.39
Reads were aligned to the human genome hg19 and to mouse genome mm10 (calibrator) using Bowtie2 and resulting SAM files were further processed and duplicates were removed using samtools v1.10
Resulting bam files were spike-in normalized and visualized using ‘bamCoverage’ of DeepTools v3.5
Raw counts of Cut&Run experiments identified ATAC-seq peaks on differential ATAC-seq peaks were retrieved using HOMER v4.11
Assembly: hg19
Supplementary files format and content: bigwig filies of the processed and normlaized files
Library strategy: Cut&Run
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| Submission date |
Aug 19, 2022 |
| Last update date |
Feb 02, 2024 |
| Contact name |
CIBIO Zippo |
| E-mail(s) |
alessio.zippo@unitn.it
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| Organization name |
University of Trento
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| Department |
Cellular, Computation and Integrative Biology (CIBIO)
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| Lab |
Chromatin Biology & Epigenetics
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| Street address |
via sommarive 9
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| City |
Trento |
| State/province |
Not Applicable |
| ZIP/Postal code |
38123 |
| Country |
Italy |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE211606 |
Oncogenic enhancers prime quiescent metastatic cells to escape NK immune surveillance by eliciting a transcriptional memory [Cut&Run] |
| GSE211610 |
Oncogenic enhancers prime quiescent metastatic cells to escape NK immune surveillance by eliciting a transcriptional memory |
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| Relations |
| BioSample |
SAMN30409835 |
| SRA |
SRX17147542 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6481373_tIMEC_H3K4me3.bw |
47.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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