 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 01, 2019 |
| Title |
Input_M2.hep.bio2 (re-analysis of GSM3137722) (ChIP-Seq) |
| Sample type |
SRA |
| |
|
| Source name |
M2.hep
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Wt
age: 2-months-old
Sex: unknown
cell type: hepatocytes
antibody: none (input)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Mice are anesthetized using isoflurane (Butler Animal Health Supply, Cat# 029405) and monitored by paw pinch. A V-shaped incision was used to reveal the abdominal cavity, the intestines moved aside, and a 22 or 24 gauge catheter (Midwest Veterinary Supply, 381423; 22 gauge for M2 animals, 24 gauge for P14 animals) was used to cannulate the venae cavae while liver perfusion media pumps into the circulation, and the portal vein is immediately severed. 37°C liver perfusion media (Invitrogen 17701-038) and then liver digest media (Invitrogen 17703-034) are perfused through the liver (45 mL each for 2-month-old animals, 25 mL each for P14 animals). Livers are dissociated with a cutting motion with cell scrapers in William’s E Medium (Sigma W4128) and strained through a 100 μm filter (BD 352360). Hepatocytes are pelleted by centrifugation at 50g for 5 min at 4°C. Isolated hepatocytes were fixed in 25 mL 1% formaldehyde in PBS for 10 min at room temperature with gentle rocking and quenched with 2.3 mL 2.5 M glycine for 5min at room temperature with gentle rocking. Fixed hepatocytes were pelleted in a swinging bucket centrifuge for 5 min at 4˚C at 50g. Fixed hepatocytes were resuspended in 10 mL (age: P14) or 20 mL (age: M2) ice-cold RSB buffer (10 mM Tris pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% NP40, protease inhibitor cocktail tablet, 0.1 mM PMSF, 1% Triton-X 100) and dounced 25 times in a Wheaton dounce on ice to lyse cytoplasmic membranes. Fixed and dounced hepatocytes were pelleted for 10 min at 4˚C at 100g and resuspended in 2 mL ice-cold AS sonication-lysis buffer (10 mM Tris-HCl, pH8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, 1 mM DTT, Roche Protease Inhibitor Cocktail (Sigma 11697498001), 0.1 mM PMSF). Hepatocytes in AS-sonication lysis buffer proceeded to sonication. P14 and M2 hepatocyte H3K27me3 (Millipore 07-449) libraries were then generated using the ThruPlex DNA-seq kit Rubicon Genomics catalog #R400428. The M2 hepatocyte H3K27me3 ChIP was previously submitted for another paper (GEO accession: GSE114198, samples: GSM3137721, GSM3137722, GSM3137723, GSM3137724).
ThruPlex DNA-seq kit Rubicon Genomics catalog #R40042
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
ChIP-seq
H3K27me3.M2.hep.bio1and2.bw
H3K27me3.M2.domains.bed
|
| Data processing |
Merging: Technical replicates (FASTQs) were concatenated before alignment
Alignment: Reads were aligned to NCBI v37/mm9 build from the UCSC Genome Browser with STAR 2.4.2a with the following parameters: --outFilterMultimapNmax 1 --alignIntronMax 1
Quality control: Alignments were filtered for uniqueness to avoid PCR duplication artifacts
Track creation: For individual biological replicate bigWigs: RPM-normalized ChIP and Input bedGraphs were generated separately with genomeCoverageBed from bedtools, then ChIP minus Input values calculated for each genomic region, then converted to bigWigs (bedgraphToBigWig tool from the UCSC Genome Browser).
Merged tracks: For merged biological replicate BGRs, data was averaged for all the biological replicates (i.e., take the individual biological replicates as BGRs, merge using bedtools unionbedg -filler "N)
A" -i BGR1 BGR2 > unionBGR, then take the unionBGR, and average the values. Note: If the unionBGR has chr start stop BGRvalue1=10 and BGRvalue2=NA, then the averaged value=10, not 5.)
Domain calls: To call enriched H3K27me3 broad domains, the genome was divided into 2kb windows with a 1kb slide. ChIP divided by input scores were calculated for each window. The top 30% percent scoring windows for each were defined ?enriched windows?. Overlapping enriched windows were merged, less enriched edges were pruned and merged again, resulting enriched domains. The algorithms for for calling enriched domains was published previously (Becker et al., Mol Cell 2017).
Genome_build: mm9
Supplementary_files_format_and_content: bigWig files represent ChIP minus Input (pooled or separate as described above). The BED files describe H3K27me3 domains
|
| |
|
| Submission date |
Aug 28, 2018 |
| Last update date |
Mar 01, 2019 |
| Contact name |
Jessica Mae Grindheim |
| E-mail(s) |
jmgrindheim@gmail.com
|
| Organization name |
UNIVERSITY OF PENNSYLVANIA
|
| Department |
Cell and Developmental Biology
|
| Lab |
Kenneth S Zaret
|
| Street address |
3400 Civic Center Blvd SCTR 9-169
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19146 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE119112 |
PRC2 proteins EZH1 and EZH2 regulate postnatal hepatic maturation [ChIP-Seq] |
| GSE119219 |
PRC2 proteins EZH1 and EZH2 regulate postnatal hepatic maturation |
|
| Relations |
| Reanalysis of |
GSM3137722 |
| BioSample |
SAMN09927021 |
| SRA |
SRX4616077 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
