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| Status |
Public on May 05, 2023 |
| Title |
HSC_QS_H3K18la_rep2 |
| Sample type |
SRA |
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| Source name |
Liver
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| Organism |
Mus musculus |
| Characteristics |
tissue: Liver
cell type: Hepatic stellate cell
genotype: Hk2 fl/fl
treatment: Untreated
antibody: H3K18la
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| Treatment protocol |
Mouse primary HSCs from Hk2 fl/fl mouse liver were transduced by adenovirus expressing either eGFP (ACT) or Cre recombinase fused to eGFP to delete Hk2 (KO), and cultured for 6 days. KO cells were treated with 10 mM lactate (LAC) for 24 h.
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| Growth protocol |
Mouse primary HSCs were isolated using density gradient centrifugation. Freshly isolated cells from mouse liver were considered quiescent HSCs (QS). To obtain activated mouse primary HSCs, cells were maintained for 6 days in Dulbecco’s modified Eagle’s medium (DMEM) containing 1,000 mg/L glucose supplemented with 10% fetal bovine serum and 1% penicillin‒streptomycin at 37 °C in a humidified atmosphere with 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Mouse primary HSCs were incubated with NE1 buffer on ice for 10 min. After collecting nuclear pellets, concanavalin A-coated magnetic beads were mixed and incubated for 10 min at RT. Next, the unbound supernatant was removed, and the bead-bound cells were resuspended in antibody buffer. Anti-H3K18la (PTM-1406, PTM Biolabs), anti-H3K18ac (ab1191, Abcam), and anti-IgG negative control (13-0042, EpiCypher) antibodies were added, and incubated on a nutator overnight at 4 °C. The primary antibodies were removed, and incubation with 0.5 ug of secondary antibody was performed on a nutator for 1 h at RT followed by incubation with pAG-Tn5 in 300-wash buffer for 1 h at RT. The bead/nucleus pellet was resuspended in tagmentation buffer, and the mixture was incubated for 1 h at 37 °C. Next, the bead/nucleus pellet was resuspended in TAPS buffer. The bead/nucleus pellet was then resuspended in SDS release buffer and incubated for 1 h at 58 °C. Next, SDS quenching buffer was added.
To amplify the libraries, 2 ul of a universal i5 and a uniquely barcoded i7 primer (10 uM stocks) were added. A volume of 25 ul of NEBNext High-Fidelity 2x PCR Master mix was added and mixed. The samples were placed in athermocycler with aheated lid using the following cycling conditions: 58 °C for 5 min; 72 °C for 5 min; 98 °C for 45 sec; 14 cycles of 98 °C for 15 sec and 60 °C for 10 sec; final extension at 72 °C for 1 min; and hold at 8 °C. Post-PCR clean-up was performed by adding 1.3x AMPure XP beads, and the libraries were incubated with the beads for 10 min at RT, washed twice gently in 80% ethanol, and eluted in 15 ul of 10 mM Tris pH 8.0. The tubes were placed on a magnetic stand, and the liquid was withdrawn into a fresh tube.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Quality check was done using FastQC v0.11.8
Sequenced reads were mapped to mm10 (UCSC) using Bowtie2 v2.4.2
Mapped reads with a MAPQ lower than 2 were filtered and sorted by samtools v1.10
Conversion of sorted bam files into bedgraph files was done using bedtools v2.30
Peaks were called using SEACR v1.3
Bigwig files were generated using deepTools v2.0
Assembly: mm10
Supplementary files format and content: bigwig files RPKM normalized by deepTools
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| Submission date |
Apr 06, 2023 |
| Last update date |
May 05, 2023 |
| Contact name |
Nissim Hay |
| E-mail(s) |
nhay@uic.edu
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| Organization name |
University of Illinois Chicago
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| Department |
BMG
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| Street address |
900 South Ashland Ave
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60607 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE229154 |
Hexokinase 2-mediated gene expression via histone lactylation is required for hepatic stellate cell activation and liver fibrosis (CUT&Tag) |
| GSE229156 |
Hexokinase 2 and lactate |
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| Relations |
| SRA |
SRX19900125 |
| BioSample |
SAMN34105256 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7156055_HSC_QS_H3K18la_rep2_RPKM.bw |
72.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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