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| Status |
Public on Mar 01, 2019 |
| Title |
P14.Wt.sexM.rep1 (srHC-seq) |
| Sample type |
SRA |
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| Source name |
P14.Wt.sexM
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| Organism |
Mus musculus |
| Characteristics |
genotype: Wt
age: postnatal day 14
Sex: male
cell type: hepatocytes
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| 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). 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). 10 μg DNA fragments were resuspended in 50 μL TE. For large fragments: 25 μL beads (0.5 volumes) were added, incubated, and beads were removed and large DNA isolated from them as described by the manufacturer. For medium fragments: 10 μL beads (0.2 volumes) were added to the supernatant from the large beads/DNA slurry, incubated, and beads were removed and medium DNA isolated from them as described by the manufacturer. For small fragments: 35 μL beads (0.7 volumes) were added to the supernatant from the medium beads/DNA slurry, incubated, and beads were removed and small DNA isolated from them as described by the manufacturer. Large fragments further sonicated to a size amenable for Illumina sequencing. Large and small fragments were prepped for sequencing with the NEBNext Ultra DNA library prep kit (NEB catalog # E7370). Libraries were sequenced as 75 bp, single-end reads on a NextSeq 500.
NEBNext Ultra DNA library prep kit (NEB catalog # E7370)
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
srHC.P14.hep.Wt.sexM.bw
srHC.P14.hep.Wt.sexMF.bw
het.P14.hep.Wt.domains.bed
int.P14.hep.Wt.domains.bed
euch.P14.hep.Wt.domains.bed
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| Data processing |
Library strategy: srHC-seq
Demultiplexing and merging: Sequences were basecalled and demultiplexed with bcl2fastq2-v2.19.0.316 (--barcode-mismatches 0 --ignore-missing-bcls --ignore-missing-filter --ignore-missing-positions --ignore-missing-controls --auto-set-to-zero-barcode-mismatches --find-adapters-with-sliding-window --adapter-stringency 0.9 --mask-short-adapter-reads 35 --minimum-trimmed-read-length 35) and technical and sequencing replicates concatenated (FASTQs)
Alignments: Reads were aligned to NCBI v37/mm9 build from the UCSC genome broser 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 biolgical replicate bigWigs: RPM-normalized large and small bedGraphs were generated separately with genomeCoverageBed from bedtools, then log2(large/small) bedGraphs were generated (to avoid divide by zero errors: a small addend was added to small bedGraphs region with values=0, equal to the size of the smallest value in that small bedGraph ), then converted to bigWigs (bedgraphToBigWig tool from the UCSC Genome Browser).
Merged track creation: 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.)
Enriched domain calling: For heterochromatin/intermediate/euchromatin domain calling, enriched heterochromatic and euchromatic domains were called with the zdomain-caller (Becker et al, Molecular Cell 2017) with the following parameters: 10kb windows, 2kb slide, 40 percentile cutoff, and a pruning value equal to the cutoff. Intermediate domains are regions not called as heterochromatic or euchromatic, or double positive het/euch domains.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig files represent log2(large/small) (If no biological replicate number, then this is the biological replicate merged version). BED9 files are domain calls (C=closed/heterochromatin, O=open/euchromatin, I=intermediate).
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| Submission date |
Aug 29, 2018 |
| Last update date |
Mar 01, 2019 |
| Contact name |
Jessica Mae Grindheim |
| E-mail(s) |
jmgrindheim@gmail.com
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| Organization name |
UNIVERSITY OF PENNSYLVANIA
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| Department |
Cell and Developmental Biology
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| Lab |
Kenneth S Zaret
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| Street address |
3400 Civic Center Blvd SCTR 9-169
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19146 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE119218 |
PRC2 proteins EZH1 and EZH2 regulate postnatal hepatic maturation [srHC-seq] |
| GSE119219 |
PRC2 proteins EZH1 and EZH2 regulate postnatal hepatic maturation |
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| Relations |
| BioSample |
SAMN09934682 |
| SRA |
SRX4623439 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3361220_srHC.P14.hep.Wt.sexM.bio1.bw |
591.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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