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| Status |
Public on Nov 27, 2024 |
| Title |
HUVEC, BRG1 CUT&RUN, PROTAC 60 minutes, 480 minutes washout, 3 |
| Sample type |
SRA |
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| Source name |
HUVEC
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| Organism |
Homo sapiens |
| Characteristics |
cell type: HUVEC
treatment: BRG1 PROTAC 60 minutes, 480 minutes washout
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| Treatment protocol |
Cells were treated at 70% confluence with 1 µM PROTAC AU-15330 or DMSO in full growth medium (EGM) for 60 min. For those with a wash out period, media was changed to normal growth medium for a subsequent 60, 240 or 480 minutes. 500,000 HUVEC were washed with wash buffer (20 mM HEPES pH 7.9, 150 mM NaCl, 500 nM spermidine, 1X Roche Protein Inhibitor Cocktail) at RT. Cells were resuspended in wash buffer and 10 µl BioMag®Plus Concanavalin A (ConA) beads (Polysciences, 86057-3) were added for 10 min at RT. Beads were separated on a magnetic rack and washed once before being resuspended in 100 µl antibody buffer (wash buffer, 0.25 % Digitonin and 2 mM EDTA) and 1 µl BRG1 antibody (Abcam, ab110641). Beads were incubated with the antibody over night with gentle shaking at 4 °C. The next day, beads were washed twice with 200 µl 0.25 % Digitonin wash buffer and resuspended in Digitonin wash buffer containing 2 µl CUTANA™ pAG-MNase (15-1016, EpiCypher, 15-1016) and incubated on ice for 30 min. Samples were washed twice and then resuspended in 100 µl Digitonin wash buffer containing 2 µl CaCl2 at a final concentration of 100 mM and incubated for 2 h at 4 °C with gentle shaking. 33 µl of 2X “stop solution” (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.25 % Digitonin, 100 µg/ml RNase A, 50 µg/ml Glycoblue) was added to the beads and incubated at 37 °C for 10 min. Samples were placed on a magnetic rack and the supernatant removed and kept for DNA purification.
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| Growth protocol |
Pooled human umbilical vein endothelial cells (HUVEC) were purchased from PromoCell (C-12203, Lot No. 405Z013, 408Z014, 416Z042, Heidelberg, Germany) and cultured at 37 °C with 5 % CO2 in a humidified incubator.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5X volume of binding buffer (20 mM HEPES pH 7.9, 20 mM KCl, 1 mM CaCl2, 1 mM MnCl2) was added to the samples and the pH adjusted with sodium acetate before being transferred to a purification column (ActiveMotif, 58002) and centrifuged at 11,000 xg for 30 sec. The column was then washed with 750 µl wash buffer and dried by centrifugation for 2 min. DNA was eluted with 25 µl elution buffer and the DNA concentration measured with a Qubit 3.0 Fluorometer (Life Technologies).
Samples were brought to 50 µl with 0.1X TE buffer and DNA end preparation performed as instructed but with incubation at 20 °C for 20 min and then 58 °C for 45 min. Adaptor ligation was performed with a 1:10 dilution of adaptor (NEB, E6440S). For DNA purification, 0.9X Volume AMPure XP beads (Beckman Coulter, A63881) was added to the samples and incubated for 5 min at RT. Beads were washed twice with 200 µl 80 % ethanol and DNA eluted with 17 µl 0.1X TE buffer for 2 min at RT. PCR amplification of the eluted DNA was performed as described in the manufacturer’s protocol but with the addition of 2.5 µl Evagreen (20X) for visualization of the amplification curves on an AriaMx Real-time PCR system (Agilent Aria 1.7). The denaturation and annealing/extension steps of the PCR amplification were performed for around 12 cycles and stopped before the curves plateaued. A cleanup of the PCR reaction was performed twice with 1.1X Ampure beads and eluted each time in 33 µl 0.1X TE buffer. DNA concentrations were measured with a Qubit (Thermo Fisher) and size distributions measured on a Bioanalyzer (Agilent).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Resulting reads were aligned to the GRCh38 genome assembly using Bowtie2 (v2.4.5) (https://doi.org/10.1038/nmeth.1923) with the parameters –local and –very-sensitive.
Mates were fixed with samtools (v1.1.0) (10.1093/gigascience/giab008) fixmate with the parameter –m set, and duplicate alignments were subsequently removed using samtools markdup with the –r flag set. Reads aligning to blacklisted regions as defined by ENCODE were removed using bedtools (2.27.1) (https://doi.org/10.1093/bioinformatics/btq033) intersect with the –v flag set.
Resulting BAM files were indexed using samtools index. Peaks were called using MACS3 (v3.0.0) (https://doi.org/10.1186/gb-2008-9-9-r137) with the parameters –scale-to-small –f BAM and –g hs.
Peaks were called using MACS3 (v3.0.0) (https://doi.org/10.1186/gb-2008-9-9-r137).
Differential peaks were also called using MACS3 by comparing read pileups between conditions using macs3 bdgdiff, normalized with the requisite total sequencing depths per condition. Peaks with a log likelihood greater than 3.84 were considered as differential.
Assembly: hg38
Supplementary files format and content: BigWig files of genomic coverage per replicate normalized by library depth.
Library strategy: CUT&RUN
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| Submission date |
Mar 20, 2024 |
| Last update date |
Nov 27, 2024 |
| Contact name |
Timothy Warwick |
| E-mail(s) |
[email protected]
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| Organization name |
Goethe Universität Frankfurt am Main
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| Department |
Institute for Cardiovascular Physiology
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| Street address |
Theodor-Stern-Kai 7
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| City |
Frankfurt am Main |
| State/province |
Hesse |
| ZIP/Postal code |
60590 |
| Country |
Germany |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE262059 |
LncRNA adapters determine SWI/SNF complex occupancy at gene regulatory elements [CutAndRun_PROTAC] |
| GSE262070 |
LncRNA adapters determine SWI/SNF complex occupancy at gene regulatory elements |
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| Relations |
| BioSample |
SAMN40558845 |
| SRA |
SRX24005746 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8156808_HUVEC_BRG1_CnR_PROTAC_480_WASH_3.bw |
53.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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