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Genome wide STAT3 binding analysis of primary human hepatocytes-derived organoids [ChIP-seq]

ABSTRACT: Genome wide STAT3 binding analysis of primary human hepatocytes-derived organoids [ChIP-seq]

PROVIDER: PRJNA949134 | ENA |

REPOSITORIES: ENA

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Genome wide STAT3 binding analysis of primary human hepatocytes-derived organoids [ChIP-seq]

Project description:To analyze the primary STAT3 binding sites induced by OSM, chromatin immunoprecipitation sequencing (ChIP-seq) was performed using an anti-human STAT3 antibody. HHOs were cultured in EM for 1 week and cultured EM (-OSM) for 24 hrs to deplete OSM before OSM induction, and then incubated with EM(+OSM) for 0.5 hr. HHOs at 0h and 0.5h after OSM induction were subjected to ChIP-seq analysis using ChIPmentation methodology with minor adaptations [Schmidl et al. PMID: 26280331]. Briefly, organoids were dissociated into single cells and fixed for 10 min, and then quenched by glycine. The lysed and sonicated chromatin was transferred to a new tube. The chromatin was reacted with the anti-STAT3 antibody (Cell Signaling Technology: Stat3 (D3Z2G) Rabbit mAb #12640) on a rotator overnight at 4°C. Washed and blocked Dynabeads Protein A/Protein G magnetic beads in RIPA-LS supplemented with 0.1% BSA were added to the lysates and incubated for 2 hr on a rotator at 4°C. Beads were subsequently washed with RIPA-LS, RIPA-HS , RIPA-LiCl, and Tris-Cl pH 8.0. The beads were then resuspended in a tagmentation reaction mix containing 1 μl of TDE1 Tagment DNA Enzyme (Illumina) and incubated at 37°C for 3 min on a thermocycler. The beads were further washed and incubated with Pronase for 1 hr at 42°C and 8 hr at 65°C to revert cross-linking. Finally, DNA was purified with MinElute columns and eluted with elution buffer. The DNA was subsequently amplified with Nextera sequencing primers and NEBNext high fidelity 2x PCR master mix for 5–10 cycles. Enriched libraries were purified, size selected using AMPure XP beads to recover libraries with the fragment length of 200–400 bp, and sequenced by llumina HiSeq 2500 sequencer with 150 bp paired-end reads.

2025-01-15 | GSE228248 | GEO

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Project description:In this study, we established human hepatocyte organoids (HHOs) that were expanded from primary human hepatocytes (PHH) in a defined medium. Briefly, the cryopreserved primary human hepatocytes (purchased) were thawn in advanced DMEM/F12 (Thermo) or Cryopreserved hepatocyte Recovery Medium (Gibco). Then, the cells were embedded in Matrigel (Corning) and cultured with the expansion medium (EM). For the eHHOs, expansion medium (EM) was prepared with a basal medium (Advanced DMEM/F12 was supplemented with penicillin/streptomycin, 10 mM HEPES, 2 mM GlutaMAX, 1 ×(Thermo Fisher Scientific), 10 nM gastrin I (Sigma), and 1 mM N-acetylcysteine (Wako, Japan) ) containing the following niche factors: 50 ng/ml mouse recombinant EGF (Thermo Fisher Scientific), 25 ng/mL human recombinant HGF (Peprotech), 100 ng/mL human recombinant FGF-10 (Peprotech), 10 uM Forskolin (Cayman Chemical), 25 ng/ml mouse recombinant noggin (Peprotech), 1 mg/ml human recombinant R-spondin1 (R; R&D), 20% Afamin-Wnt-3A serum-free conditioned medium (W; Mihara et al., 2016), 5 uM A83-01 (Tocris), and 20ng/ml human Oncostatin M (OSM; Peprotech). For dHHOs, eHHOs were cultured for 10-14 days in differentiation medium (DM), which was EM without OSM and WR, and containing a hormone cocktail (10ng/ml Growth Hormone, 10ng/ml Prolactin, and 100 ng/ml Cortisol) and 10uM DAPT (differentiation medium: DM).

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Identification of myeloid-derived growth factor as a mechanically-induced, growth-promoting angiocrine signal for human hepatocytes

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Project description:We report the genome-wide profiling of FXR binding by ChIP-seq from GW4064 or DMSO treated primary human hepatocytes. We reported altered RNA expression profiles in primary human hepatocypes upon GW4064 treatment compared to DMSO control by RNA-seq. We also reported the altered RNA expression profiles in livers from WT C57BL/6J mice upon GW4064 treatment compared to vehicle control. Primary human hepatocytes were treated with 5uM GW4064 or DMSO control, 1 hour later, cells were fixed and collected for chromatin isolation. 24 hours post treatment, cells were isolated for RNA isolation. This submission represents HTS component of study.

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