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| Status |
Public on Nov 28, 2023 |
| Title |
Isotype_18hpi, rep1 NextSeq 500 |
| Sample type |
SRA |
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| Source name |
Liver
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| Organism |
Mus musculus |
| Characteristics |
tissue: Liver
cell type: Hepatocyte
genotype: Sox4
treatment: AAV8-TBG-Sox4-P2A-Cre injected; harvested 18 hours post injection
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| Treatment protocol |
AAV8-TBG-HA-Sox4-P2A-Cre (Sox4) was retro-orbitally injected at 1e12 gc/mouse. Eighteen hours and four days post injection, hepatocytes were harvsted.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
After washing in wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl (Sigma), 0.5 mM spermidine (Sigma) and EDTA-free protease inhibitors (Roche)) twice by centrifugation at 600 ×g at 4 °C for 3 minute
The cells were resuspended in 150 ul wash buffer, 15 ul Concanavalin A-coated magnetic beads (EpiCypher) was added, and the cells/bead conjugate was bound to a magnet.
After removal of the supernatant, the cells were resuspended in 50 ul antibody reaction buffer (wash buffer with 5% digitonin and 0.5 M EDTA), 0.5 ul antibody was added, and incubated at 4 °C overnight. Following two washes in 200 ul permeabilization buffer (wash buffer with 5% digitonin), beads were resuspended in 50 ul permeabilization buffer.
Then, 2.5 ul pAG-MNase (EpiCypher) was added, and the samples were incubated at RT for 10 minutes.
While on the magnet, the supernatant was removed, and the samples were washed twice in 200 ul cold permeabilization buffer. Following resuspension in 50 ul permeabilization buffer, 1 ul 100 mM CaCl2 was added to activate MNase, and MNase digestion was performed at 4 °C for 2 hours.
The reaction was stopped by adding 33 ul STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 10 ug/ml RNase A, 50 ug/ml glycogen), and 1 ul E-Coli spike-in DNA (0.5 ng/ul) (EpiCypher) to each tube.
After incubation at 37 °C for 10 minutes, beads were bound to the magnet, and the supernatant was transferred to a new tube.
The DNA was cleaned up with the NEB Monarch kit (NEB), eluted in 12 ul elution buffer, and used as CUT&RUN DNA.
Library preparation was performed using NEBNext Ultra II End Prep kit (NEB) with a slightly modified protocol.
Briefly, after adaptor ligation, HA-Sox4 CUT&RUN DNA samples were purified with 1.75× AMPure XP beads (Beckman), while histone and IgG CUT&RUN samples were purified with 1.1× AMPure XP beads.
14-cycle PCR with index primers (NEB) was performed (initial denaturation: 1 cycle of 98 °C for 45 seconds; annealing/extension: 14 cycles of 98 °C for 15 seconds and 60 °C for 10 seconds; final extension: 72 °C for 1 minute).
Finally, the library was purified with AMPure XP beads (for HA-Sox4 0.8× followed by 1.2×; for histone and isotype control 0.9×).
For samples with adaptor contamination, further AMPure bead cleanup or gel extraction was performed.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Isotype_18hpi, rep1
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| Data processing |
Reads were aligned to the mouse genome (mm10) using Bowtie2 with options ‘--very-sensitive -X 1000 --dovetail -1’
Duplicates were removed using Picard (http://broadinstitute.github.io/picard/).
Assembly: mm10
Supplementary files format and content: The bigwig files (.bw) are generated by converting the bam files to bigwig using bamCoverage with CPM normalization.
Supplementary files format and content: Peak calling for histone PTMs was performed using MACS2 without Isotype controls as input files. Narrow peaks for H3K27ac, H3K4me1 and H3K4me3 and broad peaks for H3K27me3 were used with FDR set to 0.01. Called peaks were filtered using the mm10-blacklist.v2 file.
Supplementary files format and content: For Sox4 CUT&RUN samples, which had relatively high background, peak calling was performed using MACS2 with combined Isotype controls of the corresponding time points as input files with FDR set to 0.1. Narrow peaks were used, and the called peaks were filtered using the mm10-blacklist.v2 file.
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| Submission date |
Dec 17, 2022 |
| Last update date |
Nov 28, 2023 |
| Contact name |
Takeshi Katsuda |
| E-mail(s) |
tkatsuda412@gmail.com
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| Phone |
+1-215-746-5559
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| Organization name |
University of Pennsylvania
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| Department |
Perelman School of Medicine
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| Lab |
Gastroenterology Division
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| Street address |
421 Curie Blvd, BRB II/III Rm531
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE221223 |
Profiling Sox4 binding sites and histone post-translational modifications in adult hepatocytes (CUT&RUN I) |
| GSE221225 |
Sox4 in adult hepatocytes |
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| Relations |
| BioSample |
SAMN32298823 |
| SRA |
SRX18756928 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6856397_Isotype_18hpi_1_75PE_CPM.bw |
1.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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