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| Status |
Public on Jun 10, 2024 |
| Title |
ChIP-Seq_HA-Smc6_hTERT-RPE1_TPT |
| Sample type |
SRA |
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| Source name |
hTERT-RPE1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: hTERT-RPE1
cell type: immortalized human retina pigment epithelial cells
genotype: WT HA-Smc6 overexpressing cell
treatment: Treated with TPT (10µM) 24h prior fixation
chip antibody: Anti-HA (Invitrogen, MA-27915)
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| Treatment protocol |
Cells were harvested with trypsin-EDTA and collected by low-speed centrifugation 500 g for 5min. Cells were rinsed once with PBS, resuspended in DMEM and fixed with 1% formaldehyde (Sigma 47608) for 10 min at RT before quenching with 330 mM glycine 5min at RT and then incubated 15min on ice, and washed two further times with ice-cold PBS.
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| Growth protocol |
ChIP analysis was performed using chromatin extracted from about 5 × 10^6 HA-tagged Smc6-expressing hTERT-RPE1 cells cultured in a 10- cm diameter dish. Cells were treated either with DMSO or with 10 μM of triptolide 24 hours prior fixation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
PBS, cells were resuspended and lysed for 10 min at 4°C in 1 mL Cell Lysis Buffer (20 mM Tris-HCl (pH 8.0), 85 mM KCl, 0.5% NP-40) supplemented with EDTA-free protease inhibitor cocktail (Roche, 4693132001). The nuclei were recovered by centrifugation at 500 g for 5 min at 4°C and washed once in the same buffer. Nuclei were resuspended in 500 μL Nuclei Lysis Buffer (10 mM Tris-HCl (pH 7.5), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail) and incubated for 10 min at 4°C. Chromatin was fragmented by sonication 3 × 10 s at 60% duty cycle, with 30 sec on wet-ice between sonication cycles, using a microtip-equipped SLPe sonifier (Branson Ultrasonics ™ Sonifier™, Brookfield, USA). The sonicated samples were centrifuged at 16,000 g for 10 min. The supernatants were collected and 50 µL (1/10) was set aside as input controls. The rest (450 µL) was diluted with 1500 μl ChIP Dilution Buffer (0.01% SDS, 1.2 mM EDTA, 1.1% Triton X-100, 16.7 mM Tris-HCl (pH 8), 167 mM NaCl) supplemented with protease inhibitors and mixed with 50 μL Dynabeads™ Protein G (Invitrogen, 10009D) coupled either to 3 μg anti-HA antibody (Invitrogen, MA5-27915) in case of HA-Smc6 analysis, 5 μg anti-RPB1 (8WG16) (Covance, MMS-126R) for RNA pol II, 2 μg anti-H3pan (Diagenode, C15410324) or 4 μg anti-H3.3 (Diagenode, C15210011). After overnight incubation at 4°C on a rotating wheel, the beads were washed twice with 1 mL Cell Lysis Buffer, twice with high-salt buffer (50 mM HEPES-KOH (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), once with LiCl buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 250 mM LiCl, 1% NP-40, and 1% sodium deoxycholate) and once with TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). To elute immunoprecipitated chromatin fragments, beads were incubated for 10 min at 65°C in 400 μL freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3). DNA crosslinks were reversed by overnight incubation at 65°C with 0.6M NaCl and 80 μg proteinase K (Eurobio Scientific, GEXPRK01-I5). Samples were extracted once with Phenol – chloroform – isoamyl alcohol (Sigma, 77617), once with chloroform (Reactolab SA, P02410E16), ethanol precipitated and then resuspended in water. The input DNA samples were treated identically.
ChIP-enriched DNA were used to prepare libraries and processed with the Illumina TruSeq ChIP kit according to manufacturer specifications. Library molarity and quality were assessed with the Qubit (Thermofisher Scientific) and Tapestation (Agilent Technologies - DNA High sensitivity chip). Libraries were sequenced on a HiSeq 4000 and a NovaSeq 6000 Illumina sequencers for SR100 reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Illumina HiSeq 4000 and Illumina NovaSeq 6000
Base calling: NovaSeqControl Software v1.8.0, RTA v3 ; HiSeq Control Software v2.2.68, RTA v1.18.66 ; bcl2fastq v2.20.0
Alignment: BWA (v0.7.17) was used to align FASTQ files on GRCh38.p13 reference genome (downloaded from ENCODE) augmented with Gluc_episomal DNA sequence.
Alignment conversion and sorting: the alignment produced by BWA were converted into BAM format and sorted with samtool (v1.10)
Peak calling was done with MACS2 (v2.2.7.1) callpeak using parameters --format BAM --gsize hs --SPMR --keep-dup 1 --qvalue 0.05 --nomodel --extsize 1000.
Smc6 peak filtering: the Smc6 peaks found by MACS were filtered to exclude: 1) peaks whose fold-enrichment is below 2.5; 2) peaks in telomeric region chr5:9277-12488; 3) peaks in centromeric region chr4:49709342-49712626; 3) peaks in episomal promoter regionchr6:73520103-73522066
Smc6 peaks were further annotated with ENCODE v41 genes located within a 10kb distance of them; and manually curated.
Assembly: hg38
Supplementary files format and content: bed.gz format
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| Submission date |
Apr 30, 2023 |
| Last update date |
Jun 10, 2024 |
| Contact name |
Patrick H. Viollier |
| E-mail(s) |
Patrick.Viollier@unige.ch
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| Organization name |
University of Geneva, Faculty of Medicine / CMU
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| Department |
Dept. Microbiology and Molecular Medicine
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| Street address |
Rue Michel Servet 1
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| City |
Geneva 4 |
| ZIP/Postal code |
1211 |
| Country |
Switzerland |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE231328 |
Human Smc5/6 recognizes transcription-generated positive DNA supercoils |
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| Relations |
| BioSample |
SAMN34472139 |
| SRA |
SRX20147341 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7265145_ChIP-Seq_HA-Smc6_hTERT-RPE1_TPT.bed.gz |
130.5 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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