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| Status |
Public on Jun 18, 2024 |
| Title |
HDAC4 BJ-hTERT/HRAS day2 RepA |
| Sample type |
SRA |
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| Source name |
BJ
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| Organism |
Homo sapiens |
| Characteristics |
cell type: foreskin fibroblasts
passages: below 20
chip antibody: HDAC4 (Paroni et al, 2004 PMID 15075374 )
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| Treatment protocol |
Cells were seeded in 200 mm plates. 4-OHT was used to induce HRAS (or empty ER) for 2 days or 8 days. At the moment of the ChIP, cross-linking was performed for 15' with 1% formaldeyde (Sigma-Aldrich, Darmstadt, Germany) and then blocked with 125 mM Glycine (Sigma-Aldrich, Darmstadt, Germany).
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| Growth protocol |
BJ cells were grown in EMEM supplemented with 10% FBS, 2mM L-glutamine, penicillin–streptomycin and NEAA (Euroclone, Milan, Italy). For each ChIP, 3.2*10^6 cells were used.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were obtained with a two-step protocol (nuclei were obtained with an hypotonic buffer (20mM TRIS-HCl pH8; 85 mM KCl; 0.5% NP-40), and nuclear membrane was lysed with Lysis buffer (10mM TRIS pH7.5; 1% NP-40, 0.5% Na-deossicholate; 0.1% SDS); then lysates were clarified from sonicated nuclei (30', medium power, Bioruptor Diagenode) and histone-DNA complexes were isolated with the relative antibodies and 8ul protein A Dynabeads (ThermoFisher). Washes were performed in LOW SALT IMMUNE COMPLEX WASH BUFFER (0.1% SDS, 1% TRITON, 20mM Tris-HCl pH8, 150 mM NaCl), HIGH SALT WASH BUFFER (0.1% SDS, 1% TRITON, 2mM EDTA, 20mM Tris-HCl pH8, 500 mM NaCl), LiCl Immune Complex Wash Buffer (0.25M LiCl, 1% NP40, 0.2% Na-deossicolato, 1mM EDTA, 10Mm Tris-HCl pH8) and TE. RNA was digested with RNAseA (2μg). DNA was de-crosslinked and purified with Zymo DNA columns.
Libraries were prepared according to Drmanac et al. (Science 2010, PMID 19892942) with combinatorial probe anchor ligation chemistry generating patterned nanoarrays of self-assembling DNA nanoballs. Briefly, after End repairing, A-tailing and Ad153 index adapter ligation of 50 ng size-selected DNA, DNA was puriefied with Ampure XP beads. KAPA HiFi Hot Start Ready Mix was used for pre-PCR amlification: 95 °C 3 ', (98 °C 20s, 60 °C 15 s, 72 °C 30s) 8 cycles, 72 °C 10'. 4 °C hold thermal cycles using , the product was purified with 1X Ampure XP beads and quantified with Qubit BR ds DNA kit. For Illumina platform, Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, 5 μg of DNA were used. DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on Illumina NextSseq550 following the manufacturer's protocols. Exome capture The hybridization of BGISEQ-500 library was performed according to the Agilent SureSelect protocol with the following optimized parameters: 1000 ng purified DNA was used for hybridization, the index block and PCR block of Agilent were replaced by a corresponding Ad153_index block (one for all indexes) and Ad153_PCR block. KAPA HiFi Hot Start Ready Mix was used for Post-PCR following the 95 °C 3 min, (98 °C 20s, 60 °C 15 s, 72 °C 30s) 13 cycles, 72 °C 10 min, 4 °C hold thermal cycle. Post-PCR products were purified and single strand DNA (ssDNA) circle was performed by using adapters; T4 DNA ligae was used for ligation (NEB, Ipswich, Massachusetts). Uncirculated DNA was digested with Exo I and Exo III. The libraries were purified with 168 μL of Ampure XP beads and quantified with Qubit BR ssDNA kit. The resulting ssDNA circle is the final library. Rolling circle amplification (RCA) was performed on ssDNA with Phi29 DNA polymerase (Thermo-Fisher, Waltham, Massachusetts) to generate DNA nanoballs (DNBs).
DNBseq Nanoballs library and TruSeq ChIP Library
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
DNBSEQ-G400 |
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| Description |
HDAC4 binding at day 2
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| Data processing |
Basecalls performed using the FastQC/MultiQC programs
ChIP-seq reads were aligned to the hg38 genome assembly using bowtie2 version 2.3.4.3 with default configuration for unpaired reads
Duplicate reads were flagged using samtools but no duplicates were removed
Peak calling was performed against input sequences using EDD for H2AX ChIP (--write-log-ratios and --write-bin-scores options) and MACS2 for HDAC4, H2BK120ac and H4K16ac (--nomodel, --extsize 150 and -q 0.05 options). ChIP-seq replicates were compared using the Irreproducibility Discovery Rate (IDR) framework, with MACS2 narrow peaks as input and applying the following settings: --input-file-type narrowPeak, --rank signal.value, --output-file-type narrowPeak.
Assembly: hg38
Supplementary files format and content: Coverage tracks were generated using the bamCoverage (deepTools) tool, taking an alignment of reads as input (BAM file) and generating a coverage track (bigWig) as output, applying the following parameters: --outFileFormat bigwig --binSize 50 -p 10 --normalizeUsing RPKM
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| Submission date |
Oct 27, 2022 |
| Last update date |
Jun 18, 2024 |
| Contact name |
Emiliano Dalla |
| E-mail(s) |
emiliano.dalla@uniud.it
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| Phone |
+390432494286
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| Organization name |
Università degli Studi di Udine
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| Department |
Department of Medicine
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| Street address |
Piazzale Kolbe 4
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| City |
Udine |
| State/province |
UD |
| ZIP/Postal code |
33100 |
| Country |
Italy |
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| Platform ID |
GPL28038 |
| Series (2) |
| GSE216677 |
A HDAC4-HDAC2 complex composes an epigenetic sensor that links the DNA damage response to the senescent program by erasing H2BK120 acetylation [I] |
| GSE216862 |
A HDAC4-HDAC2 complex composes an epigenetic sensor that links the DNA damage response to the senescent program by erasing H2BK120 acetylation |
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| Relations |
| BioSample |
SAMN31483935 |
| SRA |
SRX18044469 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6685653_BJ_RAS_DAY2_HDAC4_repA_coverage.bw |
147.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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