|
Status |
Public on Dec 21, 2022 |
Title |
H3K27Ac_Infected+shield_ChIP_Rep1 |
Sample type |
SRA |
|
|
Source name |
MRC-5 cells
|
Organisms |
Homo sapiens; Human betaherpesvirus 5 |
Characteristics |
cell line: MRC-5 cells infection: HHV5 TB40r-ddFKBP strain treatment: infected +shield, 4 dpi chip antibody: Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb #8173 (Cell Signaling Technologies)
|
Treatment protocol |
For each infection, 5x10^6 MRC5 were seeded in 15 cm dishes the day before the infection. The following day, medium was replaced with 10ml of DMEM supplemented with 10% FBS without antibiotics. Confluent MRC5 were infected with HCMV TB40R-mGFP-IE-FKBP mutant at MOI of 3 in presence of 1µM Shield1 (+Shield) or Ethanol (-Shield). After 1-hour incubation at 37°C, 10 ml of media without antibiotics with Shield 1 or Ethanol were added to the plate and the cells incubated at 37°C for 23 hours. We conducted media changes at 24, 48, 72, and 96 hpi using DMEM -10% FBS- PS with either 1µM Shield1 or Ethanol. Cells were collected at 5dpi.
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Growth protocol |
HCMV MRC5 infected with TB40R-mGFP-IE-FKBP mutant were kept in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Corning), 1,000 Units/ml Penicillin-Streptomycin (Gibco), and 1uM Shiled1 or vehicle control (Ethanol) at 37°C in a humidified incubator at 5% CO2.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
After fixation, cells were lysed and sonication with a Bioruptor Plus sonication device (Diagenode). Sonication was conducted for 25 cycles, with 30 sec on/off at high intensity. ChIP was performed by incubating the sonicated sample with either Rpb1 NTD (D8L4Y) Rabbit mAb #14958 or Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb #8173 (Cell Signaling Technologies) and the protein A/G agarose beads. Beads were washed and eluted in 200μl of elution buffer (50 mM Tris-HCl at pH 8.0, 10 mM EDTA, 1.0% SDS). The eluates were de-cross-linked and the DNA purified using the Qiagen PCR purification kit. Libraries were prepared with the Illumina Chip-SEQ Kit multiplexed with IDT unique dual index barcodes. Library quality control was assessed using Bioanalyzer High Sensitivity DNA Analysis kit (Agilent) and sequenced on an Illumina NovaSEQ6000 (100bp paired-end reads).
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|
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NovaSeq 6000 |
|
|
Description |
ChIPseq
|
Data processing |
Adapters were trimmed from reads using cutadapt version 1.13 Trimmed reads were aligned to the reference genome using Bowtie2 version 2.2.6 bigWig files were created using deepTools Genome_build: hg19 Genome_build: HHV5 TB40r-ddFKBP strain (HCMV genome fa and gtf files for alignment of bigwig files are available at https://github.com/Mary-Hummel/HCMV-reference-genomes.git) Supplementary_files_format_and_content: bigWig files
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|
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Submission date |
Apr 05, 2021 |
Last update date |
Dec 21, 2022 |
Contact name |
Mary Hummel |
E-mail(s) |
m-hummel@northwestern.edu
|
Phone |
847-322-6438
|
Organization name |
Northwestern University
|
Street address |
303 E. Chicago Ave
|
City |
Chicago |
State/province |
IL |
ZIP/Postal code |
60611 |
Country |
USA |
|
|
Platform ID |
GPL29977 |
Series (1) |
GSE171518 |
Role of the HCMV immediate early proteins in controlling the HCMV and cellular epigenomes |
|
Relations |
BioSample |
SAMN18620561 |
SRA |
SRX10515122 |