|
| Status |
Public on Feb 22, 2016 |
| Title |
gH2A.x WT OIS 2 |
| Sample type |
SRA |
| |
|
| Source name |
IMR90 Lung Fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
genotype/variation: WT
treatment: 4-OHT induced H-Ras V12
ChIP: gH2A.x
antibody: Abcam, ab2893
|
| Treatment protocol |
Senescence was induced with 4-OHT (Sigma-Aldrich). For the viral transfections, IMR90 fibroblasts were infected with virus (scrambled control (SC) or MLL1 KD shRNA) at 60% confluency on 10-cm2 plates for 24 h in the presence of polybrene. Forty-eight hours following infections, cells underwent selection with puromycin to obtain completely puromycin-resistant cell populations. Senescent cells were maintained in dishes for 10 d to ensure growth termination. Senescence was determined by monitoring CDKN2A/P16 up-regulation and down-regulation of cyclin genes and by SA-β-gal (Chemicon International).
|
| Growth protocol |
IMR90 cells (obtained from Coriell Institute for Medical Research) were grown in DMEM without phenol with 10% FBS and 1% penicillin/streptomycin at 3% oxygen. The cells were generated by retrovirally infecting normal IMR90 fibroblasts with pLNCX-ER:Ras.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells in 10-cm2 dishes were fixed in 1% formaldehyde for 5 min, and fixation was quenched with addition of glycine to 125 mM for an additional 5 min. Cells were harvested by scraping from plates and were washed twice in 1× PBS before storage at −80°C. ChIP was performed as previously described (Shah et al. 2013) except that extracts were sonicated nine times for 5 min each round (30 sec of sonication with intermediate incubation of 30 sec per round) using a Bioruptor (Diagenode). All ChIPs were performed using 500 µg of extract and 2 µg of antibody per sample. Thirty microliters of Protein G Dynabeads (Invitrogen, 100.02D) was used per ChIP.
ChIP DNA was used to make sequencing libraries using NEBNext (New England Biolabs). Libraries were quantified (Kapa Biosystems), and sequencing was performed on either Hi-Seq or NextSeq platforms (50-bp, single-end reads) (Illumina).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Demultiplexing: data were demultiplexed from the raw reads using Illumina's BCL2Fastq version 2.15.
Alignment: demultiplexed data were aligned to UCSC reference genome build hg19 downloaded from iGenomes. Alignments were generated with Bowtie 2.2.4, allowing for one distinct alignment and one mismatch per seed region (-N1 -K1). All other default parameters were used.
Track creation: reads were counted to 100-bp fixed bins using featureCounts from the Subreads package version 1.4.6-p2. Normalized TPTM was used. Input was subtracted from γH2A.X, while all H3K4me3 tracks had H3 divided to account for the extensive histone loss seen in senescence.
Genome_build: hg19
Supplementary_files_format_and_content: BigWigs: bigWig files represent the distribution of H3K4me3 and gH2A.x genome-wide in 100bp contiguous, non-overlapping bins, weighted per 10 million aligned reads, and adjusted for local sonication efficiency biases by dividing by H3 or subtracting input (also weighted per 10 million aligned reads), respectively. The file H3K4me3-H3.MLL1_KD_OIS-SC_OIS.bw is the subtraction of the OIS scramble control from the OIS MLL1 knockdown track.
|
| |
|
| Submission date |
Feb 22, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregory Donahue |
| Organization name |
The University of Pennsylvania
|
| Department |
Cell & Developmental Biology
|
| Lab |
Zaret Lab
|
| Street address |
3400 Civic Center Blvd, Bldg 421
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE78141 |
MLL1 is essential for the senescence-associated secretory phenotype [ChIP-Seq] |
| GSE78142 |
MLL1 is essential for the senescence-associated secretory phenotype |
|
| Relations |
| BioSample |
SAMN04505936 |
| SRA |
SRX1595752 |
