|
| Status |
Public on Aug 15, 2014 |
| Title |
P300b-_RB1_ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
Lung Fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: IMR90 human primary lung embryo fibroblasts
cell type: contact-inhibited IMR90
infection: P300 binding mutant-infected
chip antibody: anti-RB1 (4H1) (Cell Signaling, catalog# 9309L)
|
| Treatment protocol |
1x10^8 low passage IMR90 lung fibroblasts were grown to confluence in 15 cm dishes. After 24 h, the cells were incubated with either dl312 (delta e1a), psi5 adenoviral vectors expressing WT e1a, P300 binding mutant e1a, or RB binding mutant e1a for 1 h in media with 2% serum. Cells were crosslinked 24 h post infection.
|
| Growth protocol |
IMR90 human primary lung embryo fibroblasts (ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/ml penicillin, 100 u/ml streptomycin, and 10% fetal bovine serum (FBS) at 37°C in 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
24 h post infection (p.i.), formaldehyde was added for 10 min at 37°C. After PBS washing, cross-linked cells were scraped from the plates and washed with 1 ml of PBS containing protease inhibitors (Roche). 2x10^7 Cells were resuspended in 450 ul of lysis buffer and incubated for 10 min on ice and immediately sonicated using Misonix cup-horn sonicator. 100 ul of the lysate (corresponding to 5x10^6 cells) were used for each immunoprecipitation with a given antibody (listed below); 10 ul of the lysate was used as input. After overnight reversal of crosslinking at 65°C, samples were treated with RNase A for 30 min at 37°C, proteinase K for 2 h at 56°C. DNA was subsequently purified using phenol/chloroform extraction and precipitation. DNA concentration was measured using Qubit (Invitrogen). At least 10 ng of dsDNA for both input and IP was used for library preparation according to the manufacturer's instructions (NuGen).
Sequencing libraries were constructed from 1 ng of immunoprecipitated and input DNA using the NuGen Ovation Ultralow DR Multiplex System 1-8 kit according to protocol.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Chromatin IP against RB1 in contact inhibited IMR90 24 h after infection with adeno recombinant vectors expressing e1a p300 binding mutant
|
| Data processing |
Reads were mapped to the Human (hg19) genome using Bowtie software. Only reads that aligned to a unique position in the genome with no more than two sequence mismatches were retained for further analysis. Duplicate reads that mapped to the same exact location in the genome were counted only once to reduce clonal amplification effects.
A custom algorithm executed by MATLAB was used for further processing. The genome was tiled with 50 bp windows. Each read was extended by 150 bases (so we refer to tags as the extend read counts within a bin) and was counted as one read to each window to which it partially or fully matched. The total counts of the input and ChIP samples were normalized to each other. The input sample was used to estimate the expected counts in a window; the average value for all windows was assigned to windows with zero counts.
The total counts of the input and ChIP samples were normalized to each other. The input sample was used to estimate the expected counts in a window; the average value for all windows was assigned to windows with zero counts.
We used the Poisson distribution to estimate the probability of observing the ChIP counts within a window given the expected counts in the input sample window. We considered all windows with P-values less than 1.0 x 10^ -3 to have significant peaks.
Genome_build: hg19
Supplementary_files_format_and_content: BED and wig files were generated by a custom script executed by MATLAB.Bed files contain the coordinates of the significant windows of enrichment. Alltags wig files represents the density of total IP counts. PoissP wig files represent a -logP-value Poisson distribution to estimate the probability of observing the ChIP counts within a window given the expected counts in the input sample window.
|
| |
|
| Submission date |
Jul 23, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Bing Ren |
| E-mail(s) |
biren@health.ucsd.edu
|
| Organization name |
UCSD
|
| Street address |
9500 Gilman Drive
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE59690 |
Adenovirus Small E1A Employs the Lysine Acetylases p300/CBP and Tumor Suppressor Rb to Repress Select Host Genes and Promote Productive Virus Infection [RB1_ChIP_2] |
| GSE59693 |
Adenovirus Small E1A Employs the Lysine Acetylases p300/CBP and Tumor Suppressor Rb to Repress Select Host Genes and Promote Productive Virus Infection |
|
| Relations |
| BioSample |
SAMN02934152 |
| SRA |
SRX659760 |
