|
| Status |
Public on Mar 07, 2017 |
| Title |
H2BK5Ac - TSKI4 HDAC6 sh2 |
| Sample type |
SRA |
| |
|
| Source name |
Trophoblast Stem Cells
|
| Organism |
Mus musculus |
| Characteristics |
antibody: H2BK5Ac Active Motif
knock-in: MAP3K4 K1361R
shRNA: HDAC6 shRNA
strain: 129/SvEv
|
| Growth protocol |
Cells were cultured in the absence of feeders in 30% TS media (RPMI 1640, 20% fetal bovine serum, 1% penicillin and streptomycin, 1% L-glutamine, 1% sodium pyruvate, and 100 µM β-mercaptoethanol) and 70% MEF-conditioned TS cell media. For maintenance of the stem cell state, TS medium was supplemented with FGF4 (37.5 ng/ml) and Heparin (1 mg/ml).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed in 1% paraformaldehyde for 10 minutes, harvested, and the nuclei were isolated and lysed. The nuclear lysate was sonicated and underwent immunoprecipitation using 50 µl of dynabeads (Invitrogen) coupled to 5 µl of Active Motif α-H2BK5Ac antibody per sample. DNA-protein crosslinks were reversed overnight at 65C, and samples were ribonuclease and proteinase treated before DNA was purified using Min Elute columns (Qiagen).
Preparation of libraries was performed using 50 ng of ChIP or TSWT input DNA and the KAPA HyperPrep kit with Illumina TruSeq indexed adapters. Dual SPRI size selection was performed after 18 cycles of amplification according to the manufacturer’s protocol. The average library size was ~300 bp. 12-plex libraries were sequenced (1 X 75 bp) using an Illumina NextSeq500 ranging from 7.1-8.9 X 107 reads per sample.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
alligned to mm9 reference genome
|
| Data processing |
Using Bowtie v1.1.2 software reads were mapped to the mouse mm9 genome using parameters -v2 -m1.
Aligned reads were analyzed using Easeq v1.03. Peaks were called in each IP sample relative to input using the auto-detect window size and a p-value and FDR less than 1.0x10-5.
Signal was quantified at all genes in mouse mm9 referece genome from the TSS ± 10kb. (see supplemental file)
Genome_build: mouse mm9
Supplementary_files_format_and_content: .wig files show Aligned reads used to quantify signal at the TSS of all genes. The .txt file contains the quantified H2BK5Ac signal at the TSS ± 10kb for all genes.
|
| |
|
| Submission date |
Dec 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Amy Abell |
| E-mail(s) |
anabell@memphis.edu
|
| Organization name |
The University of Memphis
|
| Department |
Biological Sciences
|
| Lab |
Abell
|
| Street address |
3774 Walker Ave
|
| City |
Memphis |
| State/province |
TN |
| ZIP/Postal code |
38152 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE92394 |
MAP3K4 Kinase Activity Controls Chromatin Remodelers for Transitions Between Epithelial and Mesenchymal Phenotypes in Trophoblast Stem Cells [ChIP-Seq] |
| GSE92426 |
MAP3K4 Kinase Activity Controls Chromatin Remodelers for Transitions Between Epithelial and Mesenchymal Phenotypes in Trophoblast Stem Cells |
|
| Relations |
| BioSample |
SAMN06141627 |
| SRA |
SRX2422798 |
