|
| Status |
Public on Feb 11, 2019 |
| Title |
CHIPseq-MCF10A Epithelial-p53shRNA-treated with Nutlin3A-CHIP for H3K27ac |
| Sample type |
SRA |
| |
|
| Source name |
Breast mammary gland
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: Nutlin3A (5 uM)
antibody: H3K27ac
|
| Treatment protocol |
To induce p53 activation, cells were treated with Nutlin-3A (5 mM final) or DMSO (vehicle control) for 6 hours.
|
| Growth protocol |
MCF10A epithelial cells were cultured in HuMEC Complete media (Gibco, #12752010) and skin fibroblast cells were cultured in DMEM media (with 10% FBS and 1% Pen/Strep, Gibco)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For RNA-Seq: Total mRNA was extracted with E.Z.N.A. Total RNA kit (Omega; R6834-02) and Poly(A)+ RNA was isolated by double selection with poly-dT beads, using 2.5 mg total RNA, which is then followed by first- and second-strand synthesis
For RNA-Seq: Libraries were prepared using NEXTflex Rapid Illumina DNA-Seq Library Prep Kit (Bioo Scientific).
For ChIP-Seq: Cells were crosslinked at 80% confluency with 1% formaldehyde for 10 min at room temperature. Crosslinking was quenched with 125mM glycine and the resulting pellet was washed twice with cold PBS and lysed. Samples were subjected to sonication with Diagenode Bioruptor Plus for 40 cycles (30 sec on/off at high setting) for shearing chromatin to 150-500 bp average size. Immunoprecipitation reactions were performed with Diagenode IP-Star Compact Atuomated System with mentioned antibodies. Immunoprecipitated DNA was reverse-crosslinked at 65°C for 4 hours and then eluted from beads, purified using SPRI beads, and used to construct sequencing libraries.
For ChIP-Seq: Libraries were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Raw fastq reads were processed for quality control using FastQC v0.11.5
Reads were aligned to the reference genome (hg19) using STAR v 2.5.3a. Genome indices for STAR were built using the parameters --runThreadN 12 --runMode genomeGenerate --sjdbOverhang 100
Indexes built in the aforementioned step were then used to align reads with STAR using the parameters --runThreadN 12 --genomeDir <genomeDir> --readFilesIn <Sample.fastq.gz> --readFilesCommand zcat --outSAMstrandField intronMotif --outFilterIntronMotifsRemoveNoncanonical --outSAMtype BAM SortedByCoordinate --outFileNamePrefix Sample. --limitBAMsortRAM 61675612266
Gene counts were calculated using HTSeq v 0.9.1 with -f bam -s no -I gene_id -m intersection-strict parameters
Genome_build: hg19
Supplementary_files_format_and_content: The count files are tab-delimited text files with first column as geneNames and second column as geneCounts
|
| |
|
| Submission date |
Feb 22, 2018 |
| Last update date |
Feb 11, 2019 |
| Contact name |
Morgan A Sammons |
| E-mail(s) |
masammons@albany.edu
|
| Organization name |
University at Albany - SUNY
|
| Department |
Biological Sciences
|
| Lab |
Sammons
|
| Street address |
1400 Washington Ave, Life Sciences 2075
|
| City |
Albany |
| State/province |
NY |
| ZIP/Postal code |
12222 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE111009 |
Cell type-dependent control of enhancer and p53 transcriptional activity by p63 |
|
| Relations |
| BioSample |
SAMN08581053 |
| SRA |
SRX3734284 |
