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| Status |
Public on Jun 24, 2024 |
| Title |
Cut& Run on SU-DIPG13 treated with Vorinostat using H3K27ac ab |
| Sample type |
SRA |
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| Source name |
SU-DIPG13
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| Organism |
Homo sapiens |
| Characteristics |
cell line: SU-DIPG13
cell type: Glioma
cell line: SU-DIPG13
treatment: Vorinostat
time: 72 hours
genotype: H3.3K27M
antibody: anti-histone H3K27ac
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| Treatment protocol |
Cells were plated at a density of 2500 cells per well in 96-well plates in at least triplicates and subjected to drug treatment for 3 days (1uM Vorinostat), 8 days (10uM Sulfopin), Vorinostat+Sulfopin (72h,1uM+8,10uM) or DMSO. When treated for 8 days one pulse were given at day 4. Cell viability was measured by CelltiterGlo assay (G7571, Promega) according to the manufacturer’s instructions. Luminescence was measured by Cytation 5 plate reader and viability was compared to DMSO treated control cells.
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| Growth protocol |
DIPG derived cells SU-DIPG-13 (H3.3-K27M, female) were generated in the lab of Dr. Michelle Monje, Stanford University (Grasso et al., 2015). Cells were maintained at 37°C with 5% CO2 and were cultured in Tumor Stem Media (TSM) consisting of a 1:1 mixture of DMEM/F12 (Invitrogen, 31330038) and Neurobasal (-A) (Invitrogen, 10888022), with addition of HEPES Buffer Solution (1M) (Invitrogen 15630-080), MEM Sodium Pyruvate Solution 100mM (100X), liquid (Invitrogen, 11360-070) MEM Non-Essential Amino Acids Solution 10mM (100X), Invitrogen, 11140-050), GlutaMAX-I (Invitrogen, 35050-061) Antibiotic-Antimycotic (100X) (Invitrogen,15240-096) B27(-A) (Invitrogen, 12587010), human-bFGF (20 ng/mL) (Shenandoah Biotechnology, 100-146), human-EGF (20 ng/mL) (Shenandoah Biotechnology, 100-26), human PDGF-AA (20 ng/mL) (Shenandoah Biotechnology, 100-16), human PDGF-BB (20 ng/mL, 100-18) (Shenandoah Biotechnology) and heparin (10 ng/mL) (Stemcell Technologies, 07980).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cut&Run assay was done as described in (Meers et al., 2019; Skene and Henikoff, 2017) with slight modifications as follows: Cells were harvested and counted, with 200,000 cells taken per reaction. Permeabilized cells, bound to Concanavalin A-coated beads (Bangs Laboratories, BP531), were mixed with individual primary antibody (Table S1) and incubated overnight at 4°C while rotating. Secondary antibody, anti-rabbit HRP (Table S1), was used as a negative control. pAG-MNase enzyme (generated in the Department of Life Sciences Core Facilities, WIS, using Addgene plasmid 123461) was added to each sample followed by incubation step of 1 h at 4°C. Targeted digestion was done by 15 min incubation on ice block (0°C) under low salt conditions. DNA purification was done using Nucleospin gel and PCR clean-up kit (Machery-Nagel, 740609).
Libraries were prepared from 1-20ng of DNA as previously described (Blecher-Gonen et al., 2013). Briefly, DNA Fragments were repaired by T4 DNA polymerase (NEB, M0203), and T4 polynucleotide kinase (T4 PNK, NEB, M0201) was used to add a phosphate group at the 5′ ends. An adenosine base was then added by Klenow fragments (NEB, M0212) to allow efficient ligation, using T4 quick ligase (NEB, M2200), of sequencing adapters, which contain a T-overhang. DNA fragments were amplified by PCR reaction (Pfu Ultra II fusion, Agilent Technologies, 600670), which also introduced the Illumina-P5 adapter at one end of the molecule. SPRI beads were used to purify proper sized DNA fragments after each enzymatic step. Libraries were quantified by Qubit (ThermoFisher Scientific) and proper fragment range (200-400bp) was verified by TapeStation (Agilent). Sequencing was done on a Next-Seq 500 instrument (Illumina, #20024904) using a V2 150 cycles mid output kit (paired end sequencing).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Paiered-end reads were preprocessed with cutadapt to remove adapters and low-quality bases (parameters: --times 2 -q 30 -m 20). Reads were mapped to human genome (hg38, UCSC) using Bowtie version 2.3.5.1 (--local --very-sensitive-local --no-unal --no-mixed --no-discordant -I 10 -X 700 --dovetail ).
Nucleosome fragments at the length >120bp were selected from the remaining unique reads using picard-tools, and broad peaks were called using MACS2 against the corresponding HRP samples as background control (parameters: -f BAMPE --SPMR --nomodel --extsize 100 --keep-dup auto -q 0.05). Bigwig files were constructed from BAM alignments using deepTools2 suite with bamCoverage, using RPKM-normalization in 10bp bins.
Assembly: hg38
Supplementary files format and content: Bigwig files were constructed from BAM alignments using deepTools2 suite with bamCoverage, using RPKM-normalization in 10bp bins.
Supplementary files format and content: Peaks files were constructed using MACS2 against HRP samples as background control.
Library strategy: CUT&RUN
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| Submission date |
Dec 22, 2022 |
| Last update date |
Jun 24, 2024 |
| Contact name |
Danielle Algranati |
| E-mail(s) |
Danielle.algranati@weizmann.ac.il
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| Organization name |
Weizmann Institute of Science
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| Department |
Department of Immunology & Regenerative Biology
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| Lab |
Efrat Shema
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| Street address |
234 Herzl Street
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| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE221611 |
Cut&Run SU-DIPG13 cells treated Sulfopin/Vorinostat/Sulfopin+Vorinostat or DMSO |
| GSE221614 |
Cut&Run and MARS-seq on SU-DIPG13 cells treated Sulfopin/Vorinostat/Sulfopin+Vorinostat or DMSO |
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| Relations |
| BioSample |
SAMN32372447 |
| SRA |
SRX18827400 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6890295_Vorino_27ac_S25_RPKM.bw |
118.1 Mb |
(ftp)(http) |
BW |
| GSM6890295_Vorino_27ac_S25_vs_DIPG13-HRP_peaks.broadPeak.gz |
331.8 Kb |
(ftp)(http) |
BROADPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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