 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 26, 2021 |
| Title |
E_STAT1_4h_LipidA_BI9564_ChIPseq |
| Sample type |
SRA |
| |
|
| Source name |
C57BL/6, Bone-marrow derived macrophages (BMDMs)
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
treatment: Bi9564 and Lipid A for 4h
cell type: Bone-marrow derived macrophages (BMDMs)
shRNA: none
chip antibody source: Cell Signaling 91728
|
| Treatment protocol |
On day 7, BMDMs were pre-treated overnight with 500 nM JQ1 (Tocris 4499); 3 uM dBRD9 (Tocris 6606); 3 uM BI9564 (Tocris 5590); and 10 uM I-BRD9 (Tocris 5591). On day 8, BMDMs were treated with vehicle or 100ng/ml Lipid A.
|
| Growth protocol |
Bone marrow was harvested from the femurs and tibias of 8 to 12 week-old C57BL/6 mice. Bones were crushed and bone marrow progenitors were isolated and cultured in RPMI-1640 supplemented with 30% M-CSF, 20% fetal bovine serum (FBS), 10mM HEPES, 1mM sodium pyruvate, MEM non-essential amino acids, and penicillin/streptomycin on petri dishes. On day 6, BMDMs were lifted using cold PBS and replated onto tissue-culture treated dishes in half-M-CSF media and half Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. RAW 264.7 cell line was cultured in DMEM supplemented with 10% FBS.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA was isolated using Quick-RNA Miniprep Kit (Zymo Research). For ChIP-Seq, 15 million BMDMs were collected and cross-linked first in 3mM disuccinimidyl glutarate (DSG) then in 1% formaldehyde. After quenching the excess formaldehyde with 125 mM glycine, the fixed cells were washed, pelleted and flash-frozen. Upon thawing, the cells were resuspended in lysis solution (50 mM HEPES-KOH pH 8, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100 and incubated on ice for ten minutes. The isolated nuclei were washed with wash solution (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl) and shearing buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris-HCl pH 8) then sheared in a Covaris E229 sonicator for 10 minutes to generate DNA fragments between ~ 200-1000 bp. After clarification of insoluble material by centrifugation, the chromatin was immunoprecipitated overnight at 4C with antibodies against BRD9, BRD4, IRF9, STAT1, or STAT2. The next day, the antibody bound DNA was incubated with Protein A+G Dynabeads (Invitrogen) in ChIP buffer (50 mM HEPES-KOH pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate). Antibody bound DNA were washed and treated with Proteinase K and RNase A and the purified ChIP DNA was used for library generation for subsequent sequencing.
ChIP-Seq libraries were generated using the NuGen Ovation Ultralow Library System V2 following the manufacturer's instructions. RNA-Seq libraries were prepared using either Illumina TruSeq RNA Library Prep Kit v2 or Illumina TruSeq Stranded mRNA Kit following the manufacturer's instructions.
ChIP-Seq and strand-specific polyA+ RNA-seq
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
ChIPseq of STAT1 4h Lipid A BI9564-treated BMDMs
|
| Data processing |
Basecalls performed using RTA (Illumina)
Reads were aligned to the mouse genome (mm10) using STAR alignment tool (v2.5) for RNA-seq and ChIP-seq. In all cases, only reads that mapped to a unique genomic locations (MAPQ > 10) were used for downstream analysis.
HOMER (v4.8) (http://homer.salk.edu/homer/) was used to process alignment files to generate normalized bedGraphs for the UCSC Genome Browser. ChIP-seq peaks for BRD9, BRD4, BRD2, IRF9, STAT1, and STAT2 were found by using the findPeaks program in HOMER with the default parameters for option "-style factor" using the appropriate ChIP input experiments as background. For RNA-Seq, Reads Per Kilobase of exon per milion mapped reads were calculated using HOMER (http://homer.salk.edu/homer/) across annotated gene exons (RefSeq).
Genome_build: mm10
Supplementary_files_format_and_content: bedGraph file Columns: (1) chrom (2) chromStart (3) chromEnd (4) dataValue. DESeq Tab delimited text file: for DESeq_0hIFNa_Vehicle.txt: Columns: bedGraph file Columns: (1) chrom (2) chromStart (3) chromEnd (4) dataValue. DESeq Tab delimited text file: for DESeq_0hIFNa_Vehicle.txt: Columns: (1) RefSeq acc (2) chr (3) start (4) end (5) strand (6) length (7) copies (8) annotation (9-12) normalized Log2 read count for experiment (13) Log2 fold change (14) p-value, adjusted (15) p-value; for DESeq_4hIFNa_Vehicle_dBRD9_IBRD9_JQ1.txt bedGraph file Columns: (1) chrom (2) chromStart (3) chromEnd (4) dataValue. DESeq Tab delimited text file: for DESeq_0hIFNa_Vehicle.txt: Columns: (1) RefSeq acc (2) chr (3) start (4) end (5) strand (6) length (7) copies (8) annotation (9-16) normalized Log2 read count for experiment (17,18,19) Log2 fold change, p-value, adjusted p-value dBRD9 4h IFNa vs vehicle 4h IFNa (20,21,22) Log2 fold change, p-value, adjusted p-value of IBRD9 4h IFNa vs vehicle 4h IFNa (23,24,25) Log2 fold change, p-value, adjusted p-value of JQ1 4h IFNa vs vehicle 4h IFNa
|
| |
|
| Submission date |
Nov 23, 2021 |
| Last update date |
Nov 17, 2022 |
| Contact name |
Diana C. Hargreaves |
| E-mail(s) |
[email protected]
|
| Organization name |
The Salk Institute for Biological Studies
|
| Street address |
10010 North Torrey Pines Road
|
| City |
San Diego |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE176146 |
Bromodomain protein 9 (BRD9) regulates interferon-stimulated genes during macrophage activation via cooperation with BET protein BRD4 |
|
| Relations |
| BioSample |
SAMN23404521 |
| SRA |
SRX13205879 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5700432_E_STAT1_BI9564_4hLipidA.ucsc.bedGraph.gz |
163.9 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
