|
| Status |
Public on Dec 21, 2022 |
| Title |
PolII_Infected-shield_ChIP_Rep2 |
| 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 no shield 4 dpi
chip antibody: Rpb1 NTD (D8L4Y) Rabbit mAb #14958 (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.
|
| 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).
|
| |
|
| 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
|
| |
|
| 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 |
SAMN18620549 |
| SRA |
SRX10515135 |
