|
| Status |
Public on Nov 17, 2018 |
| Title |
Control_2 |
| Sample type |
SRA |
| |
|
| Source name |
Control_2
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: control
cell type: in vitro cultured GC B cells
chip antibody: STAT3 Ab (124H6, Cell Signaling Technology)
|
| Growth protocol |
C57BL/6 T and/or B cell STAT3 conditional knockout (cKO) mice (STAT3fl/flCD2Cre/+ and STAT3fl/flCD19Cre/+) were generated by interbreeding STAT3fl/flCD2+/+CD19+/+ mice 15 (control) with STAT3+/+CD2Cre/Cre or STAT3+/+CD19Cre/Cre mice (The Jackson Laboratory), respectively. Mice at 6–8 weeks of age with both sexes were used for experiments. All mice were housed and bred in a conventional facility at the University of Louisville. Animal care and experiments were conducted in accordance with the National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee at the University of Louisville
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
In vitro cultured GC B cells from STAT3 control and cKO mice were used for ChIP studies as described previously (Tripathi et al. 2017. Cell Reports). In brief, cells were subjected to sonication using a sonicator (AFA Focused ultrasonicator S220) to obtain chromatin fragements of 100-500 bp. Fragemented chromatin was incubated with STAT3 Ab (124H6, Cell Signaling Technology) or isotype control Ab and incubated for crosslinking with the beads (Dynabeads Protein G, Invitrogen). After crosslinking, crosslinks were reversed (65C for 12-16 h), and precipitated DNA was treated with Proteinase K and then purified (QIAquick PCR purification kit, Qiagen).
DNA was DNA-end repaired, with a 3'-dA overhang and ligation of methylated sequencing adaptor. Sequences were PCR amplified and size selected in the range of 100-500bp (including adapter sequence). Selected sequences were then prepared using the BGISEQ-500 ChIP-Seq sample preparation workflow.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
BGISEQ-500 |
| |
|
| Data processing |
Raw reads were filtered by BGI to remove low quality reads, N reads, and adaptor sequences to obtain clean data. Raw reads were removed if: 1) The ratio of N in whole read was over 10%; 2) unknown bases are more than 10%; or 3) The ratio of base whose quality was less than 20 was over 10%.
The resulting clean data was algined to the mm10 reference genome using SOAPaligner/SOAP2 (Version: 2.21t) allowing no more than 2 mismatches.
Peak calling was performed using using MACS (Model-based Analysis for ChIP -Seq, version: MACS-1.4.2) with a p-value cutoff of 0.05. The parameters used were: macs2 -g hs -s 50 -p 1e-5 -m 10 30 --broad -B --trackline
Genome_build: mm10
Supplementary_files_format_and_content: wig files were generated using the bam2wig program from samtools and represent the mapped read locations
|
| |
|
| Submission date |
Sep 17, 2018 |
| Last update date |
Nov 17, 2018 |
| Contact name |
Eric Christian Rouchka |
| E-mail(s) |
eric.rouchka@louisville.edu
|
| Organization name |
University of Louisville
|
| Department |
Biochemistry and Molecular Genetics
|
| Lab |
KY INBRE Bioinformatics Core
|
| Street address |
522 East Gray Street
|
| City |
Louisville |
| State/province |
Kentucky |
| ZIP/Postal code |
40292 |
| Country |
USA |
| |
|
| Platform ID |
GPL23479 |
| Series (1) |
| GSE120022 |
STAT3 binding to DNA in wildtype vs B cell specific STAT3 knockout mice |
|
| Relations |
| BioSample |
SAMN10077401 |
| SRA |
SRX4702345 |
