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| Status |
Public on Mar 04, 2024 |
| Title |
ATAC of HEK293 TNFa with TRAFTAC against NfkB, rep2 |
| Sample type |
SRA |
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| Source name |
HEK293
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| Organism |
Mus musculus |
| Characteristics |
cell line: HEK293
cell type: HEK293
knockdown: NFkB
treatment: TNF-alpha activation
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| Treatment protocol |
For the transfection cells at a confluency of 70-90% were incubated for 6h with 1 ug/mL of a plasmid encoding N-terminal HA-HaloTag7-containing dCas9 fusion protein and 25 nM TRAFTAC using Lipofectamine 3000 following the suppliers' instructions. The plasmid used was published and kindly provided by the Crews lab (Samarasinghe et al., 2021). Cells were then incubated for 19h with 5 ng/mL of TNF-alpha (Sigma-Aldrich, H8916) and 25 uM HP14 (dissolved in DMSO) (WuXiAppTec) respectively DMSO.
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| Growth protocol |
HEK293 cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) with 10% Fetal Bovine serum (FBS) (Gibco,10500064) and 1% Penicillin/Streptomycin (P/S) (Gibco,15140122). All of the incubation steps were performed at 37 Celsius and 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
30 min before harvest, cells were treated with 200 units/ml DNase (Sigma, DN25). HEK293 cells were then washed twice with PBS (Gibco, 10010023) and detached with TrypLE (Gibco, 12605010). Enzymatic digestion was quenched with growth medium and cells were washed once again with PBS.
ATAC libraries were prepared from 50 000 cells per sample according to the published OmniATAC protocol (Corces et al., 2017) with minor adaptations. In short, samples were washed with cold 50 ul of PBS, afterwards cells were incubated in 50 ul lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin) for 3 min on ice. Cells were washed with 1 ml of lysis buffer without Digitonin and NP-40. Transposition was done in 50 ul reaction buffer (1x tagmentation buffer (Diagenode, C01019043), 0.1% Tween-20, 0.01% and 2.5 ul Tn5 (Diagenode, C01070012)) for 30 min at 37 Celsius and 1000 RPM shaking. Subsequent DNA purification was performed with MinElute Reaction Cleanup Kit (Qiagen, 28204). PCR amplification was done with NEBNext High Fidelity 2X PCR Master mix (NEB, M0541) and UDI primer (Diagenode, #C01011034) with concentrations according to manufacturer's recommendation. Total number of needed amplification cycles (8x) was controlled with qPCR. Library purification and fragment size selection was done with AMPure XP beads (Beckmann, A63880). 20 ul of 10 nM was sent for sequencing externally with Novogene (Cambridge, UK). A minimum of 20 Mio PE reads were sequenced per sample.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
peaks_fromMergedSamples.narrowPeak
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| Data processing |
Adapters were removed with trimmomatic before alignment on GRCh38.p10 using bowtie2 with --dovetail --no-mixed --no-discordant -I 15 -X 2000. Duplicates were marked with picard.
Peaks were called on the merge of reads from all samples using MACS2.
Bigwig files of (shifted) insertion sites (normalized to library size) were generated using the epiwraps R package.
Assembly: GRCh38
Supplementary files format and content: Peaks (called on the merge of all samples), per-sample bigwig files of insertion sites
Supplementary files format and content: peakCounts.SE.rds.gz is a R object containing a SummarizedExperiment of the peak counts.
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| Submission date |
Feb 28, 2024 |
| Last update date |
Mar 04, 2024 |
| Contact name |
Pierre-Luc Germain |
| Organization name |
ETH Zürich
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| Department |
D-HEST Institute for Neuroscience
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| Street address |
Winterthurerstrasse 190
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| City |
Zürich |
| ZIP/Postal code |
8057 Zürich |
| Country |
Switzerland |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE260504 |
ATAC-seq of TNF-alpha activation in HEK cells with or without TRAFTAC against NfkB |
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| Relations |
| BioSample |
SAMN40191529 |
| SRA |
SRX23784534 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8118541_NFkB_PG_2.inserts.bw |
208.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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