 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 08, 2016 |
| Title |
FoxA3 ChIP-seq in FoxAflox;Alfp-Cre Mice |
| Sample type |
SRA |
| |
|
| Source name |
Liver
|
| Organism |
Mus musculus |
| Characteristics |
strain: B6
background: FoxAflox;Alfp-Cre
replicate: 1
ChIP: FoxA3
antibody vendor/catalog: Santa Cruz, sc-5361
|
| Treatment protocol |
Mouse liver was perfused with 10 ml of 50 ng/ml heparin in PBS, followed by 5 ml 1% formaldehyde in PBS. Perfused livers sat for 10 min including formaldehyde perfusion time at room temperature. The mouse was then sacrificed by cervical dislocation. The livers were removed and placed in 6 ml Buffer A (15 mM Hepes [pH 7.6], 60 mM KCl, 15 mM NaCl, 0.2 mM EDTA, 0.5 mM EGTA, 0.34 M sucrose, 0.15 mM 2-mercaptoethanol) supplied with 125 mM glycine, and were minced with scalpels on ice. The liver was homogenized with a Teflon pestle in a glass homogenizer. The nuclear suspension was filtered through a 100 mm cell strainer.
|
| Growth protocol |
Mice were anesthetized in a chamber with isoflurane (Butler Animal Health Supply). When the mouse was completely under anesthetic, it was transferred to a surgical surface and its head was inserted into a nose cone containing isoflurane.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The nuclear suspension was layered onto a 1.5 ml cushion of 1:1 of buffer A: buffer B (15 mM Hepes [pH 7.6], 60 mM KCl, 15 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 2.1 M sucrose, 0.15 mM 2-mercaptoethanol) in 15 ml Corex tubes, centrifuged at 7.5-10K rpm for 10 min at 4°C, and the supernatant was removed. The pellets were suspended in 5-10 ml Buffer A and layered again onto 1:1 of buffer A:B, and centrifuged at 5K rpm for 10 min at 4°C, and the supernatant was removed. The pellet was suspended into 4 ml sonication buffer (50 mM Tris [pH8], 2 mM EDTA, 0.5 % N-Lauroylsarcosine, complete EDTA-free protease inhibitor cocktail (Roche), 0.5 mM PMSF) for Bioruptor sonication, or about 8 ml sonication buffer as adjusted the concentration at 2-2.4 x 107 nuclei/ml for Covaris sonication, and incubated for 5 min at room temperature, then 5 min on ice. The nuclear lysate was transferred to milliTUBE ATA Fiber tubes (#520130) and sonicated for 8 min (Temperature: 5-9 C, PP: 200, DF 10, CB 200) using Covaris S220. A portion of chromatin was reverse-crosslinked and confirmed, by agarose gel electrophoresis, that their average DNA fragment size was around 200 bp. RNase A was added to 12.5-25 ng/ul and incubated at room temperature for 10 min. Debris was removed by centrifugation at 14K rpm for 10 min at 4°C. The supernatant was dialyzed in TE (10 mM Tris [pH8], 1 mM EDTA) using 3500 MWCO Slide-A Lyzer Dialysis cassette (Thermo Scientific) at 4°C for several hours, and move into new TE and dialyzed overnight. 20 ul (for H1 ChIP) or 50 ul (for FoxA3, C/EBPβ, and HNF4α ChIP) Dynabeads Protein G (Life technologies) were washed three times with 500 ul blocking solution (0.5 % w/v BSA in PBS). The beads were resuspended in 20 ul or 50 ul blocking solution and saturated with 2.5 ug H1 antibody (Millipore, 05-457), 5 ug FoxA3 antibody (Santa Cruz, sc-5361), 5 ug C/EBPβ antibody (Santa Cruz, sc-150), 5 ug HNF4α antibody (Abcam, ab42898) by rotating the mixture for 2-6 hr at 4°C. The antibody-beads were washed two times with 500 ul blocking solution and two times with 500 ul ChIP buffer (50 mM Hepes-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 0.1 % Triton X-100, 1 mM PMSF, Complete EDTA free protease inhibitor cocktail (Roche)). ChIP was performed in 100 ul (for H1) or 250 ul (for FoxA3, C/EBPβ, and HNF4α) ChIP buffer containing antibody-beads mixture and 4-12.5 ug (for H1) or 50 ug (for FoxA3, C/EBPβ, and HNF4α) of sonicated chromatin, which was based on O.D. A260 of reverse-crosslinking DNA, and rotated for 16 hr at 4°C. The ChIP-beads mixture was washed for 5 min with rotation successively two times with 500 ul ChIP buffer, one time with 500 ul ChIP buffer plus 500 mM NaCl, two times with 500 ul LiCl solution (10 mM Tris-HCl [pH 8], 250 mM LiCl, 1 mM EDTA, 0.5% NP-40), and two times with 500 ul TE plus 0.1% NP-40. The chromatin was eluted from the beads in 67 ul TES (50 mM Tris-HCl [pH 8], 10 mM EDTA, 1% SDS) and incubated at 65°C for 15 min. The supernatant was placed in a fresh tube and the elution was repeated two more times, combining all supernatants. The eluted chromatin and input chromatin were reverse-crosslinked by adding NaCl (final 200 mM) and incubating at 65°C for 4 to 16 hr. The chromatin was treated with 0.2 mg/ml Proteinase K and incubated at 37°C for 2 hr. DNA was purified by phenol-chloroform extraction followed by ethanol precipitation.
We prepared multiplexed libraries from two biological replicates of H1, FoxA3, C/EBPβ and HNF4α ChIP and Input from FoxA1/FoxA2 flox;Alfp-Cre and WT. We used NEBNext Ultra DNA Library Prep Kit for Illumina (#E7370). Libraries from FoxA1/FoxA2 flox;Alfp-Cre and WT were sequenced as 75-bp single-end, using Illumina NextSeq500.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Demultiplexing: data were demultiplexed using Illumina's BaseSpace online utilities.
Alignment: data were aligned to genomic build mm8 using bowtie 0.12.7 (-m 1 --best).
Track generation: track files (bigWigs) were created by (i) pooling aligned tags from replicate data, (ii) making coverage maps of pooled tags using BEDTools' genomeCoverageBed utility (option -bg to produce bedGraphs), then (iii) multiplying these by the RPM coefficient (inverse of the number of millions of tags aligned), and finally (iv) generating bigWigs with UCSC Genome Browser's bedGraphToBigWig utility.
Genome_build: mm8
Supplementary_files_format_and_content: BigWig files describing RPM-adjusted genomic tracks with RPM-adjusted input subtracted are provided for visualization. These files represent two pooled replicates each.
|
| |
|
| Submission date |
Feb 08, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregory Donahue |
| Organization name |
The University of Pennsylvania
|
| Department |
Cell & Developmental Biology
|
| Lab |
Zaret Lab
|
| Street address |
3400 Civic Center Blvd, Bldg 421
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE57559 |
Hypersensitive Nucleosomes in Chromatin Are Intrinsic to the Structure of Active, Tissue-Specific Enhancers |
| GSE77670 |
Pioneer transcription factor FoxA maintains an accessible nucleosome configuration at enhancers for tissue-specific gene activation [ChIP-seq] |
|
| Relations |
| BioSample |
SAMN04479821 |
| SRA |
SRX1564902 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2055884_FoxA3-I.Mut.Pool.bw |
592.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
