H3k9me3 antibody

ChIP assays were conducted using the EZ-ChIP^TM kit (17–371, Millipore, CA, USA) following the manufacturer’s instructions. Briefly, after sheared by sonication, the 200–1000 bps DNA fragments were immunoprecipitaed by MINA53 antibody (397300, Thermo Fisher, IL, USA) or H3K9me3 antibody (ab113754, Abcam, MA, USA), and then reverse cross-linked and purified. qPCR was conducted subsequently to determine the enrichment flod. The primer sequences used are listed in Supplementary Table S2.

HepG2 and SMMC-7721 cell lines transfected with HBx-expressing plasmid (2 biological replicates per cell lines) were chemically crosslinked, resuspended in lysis buffer, and sonicated fragments ranged in size from 200 to 1000 bp (Supplementary Figure S1A). Sonicated chromatin was resuspended in IP buffer and incubated with magnetic beads conjugated H3K9me3 antibody (Abcam, Cambridge, UK). An un-enriched sample of DNA was treated in a similar manner to serve as input. The immunoprecipitated DNA was tested for enrichment of control loci by qPCR validation (primers and results are listed in Supplementary Table S6 and Supplementary Figure S1B, respectively). IgG control levels were below the threshold. The purified ChIP and input DNA was amplified and then fluorescently labeled using the NimbleGen Dual-Color DNA Labeling Kit, which is a single array designs that includes 23,148 gene promoter regions (from about -1300 bp to +500 bp of TSSs) totally covered by ~180,000 probes with approximately 210 bp spacing, dependent on the sequence composition of the region. The Cy5-ChIP and Cy3-input labelled DNA samples were co-hybridized to ArrayStar Human RefSeq Promoter Arrays, post hybridization washes were carried out and microarrays were scanned using Agilent Scanner G2505C.

The antibody used for western blot analysis are listed as follows: JMJD2D antibody (Abcam, Cat# ab93694, 1:1000); β-actin antibody (Sigma Aldrich, Cat# A5441, 1:1000); EpCAM antibody (Cell Signaling Technology, Cat# 93790, 1:1000); β-catenin (Cell Signaling Technology, Cat# 8480, 1:1000); TCF4 (Cell Signaling Technology, Cat# 2569, 1:1000); Flag-tag antibody (Sigma, Cat# F1804, 1:1000); Myc-tag antibody (ABclonal, Cat# AE070, 1:1000); H3K9me3 antibody (Abcam, Cat# ab8898, 1:1000); Sox9 (Abcam, Cat# ab182579, 1:1000); Notch1 (Cell Signaling Technology, Cat# 3608, 1:1000); HA-tag antibody (Thermo Fisher Scientific, Cat# 26183, 1:1000). The JMJD2D inhibitor 5-c-8HQ (HY-12304) was purchased from MCE. Lipofectamine 2000 (Cat #11668019) was purchased from Invitrogen.

Cells were cultured in coverslips (15 mm diameter) in a 12-well plate. The next day, cells were washed with PBS and, after that, fixed in PBS containing 4% v/v PFA for 15 min at RT, washed three times with PBS, permeabilized in PBS containing 0.3% v/v Triton for 10 min, and blocked in blocking solution (3% BSA in 0.3% Triton-PBS) for 30 min. Coverslips were then incubated for one hour in a humid chamber with primary antibody diluted 1:150 in blocking solution (H3K9me3 antibody (Abcam, ab8898), BRG1 antibody (A300-813A, Bethyl), and Emerin antibody (10351-1-AP, Proteintech)). Coverslips were washed four times with PBS and incubated with 140 ng of ProtA-Turbo enzyme diluted in 30 μl of blocking buffer per coverslip for one hour and then washed 4 times with PBS. Coverslips were then incubated with biotin reaction buffer (5 mM MgCl₂, 5 μM Biotin, 1 mM ATP in PBS) for 10 min at 37 °C, followed by three washes with PBS. Finally, coverslips were incubated with secondary antibody (anti-rabbit Alexa fluor 568 (Life Technologies A11004, dilution 1:1000), FITC-Avidin (ThermoFisher Scientific A821, 1:200) and DAPI for one hour, washed 4 times with PBS and fluoromount G (Thermo, 00-4958-02) was used for mounting the slides. Images were acquired with a confocal microscope LSM900 (ZEISS) and analyzed with Fiji software.

Chromatin Immunoprecipitation (ChIP) was performed by the modified protocol from Simple ChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads, #9005). Briefly, the mycelia were fixed with 1% formaldehyde for 15 min at 45 °C with shaking and then stopped with glycine at a final concentration of 125 mM. Cross-linked tissues were ground and resuspended at 1 g/mL in lysis buffer containing 1 mM PMSF, 1 μg/ml pepstatin A and 1 μg/mL leupeptin. Chromatin was sheared by sonication to 100–1000-bp fragments. The chromatin immunoprecipitation was performed using 4 μg H3K9me3 antibody (ab8898, abcam) per 2 mg protein. After DNA extraction, the pellets were resuspended in 300 μL of DNase-free water.
For ChIP-seq, library construction was performed by E-GENE Corporation (Shenzhen, China), and pair-end sequencing was performed on Illumina NovaSeq6000 platform. After pass quality control with Trim Galore (version 3.2), paired-reads were mapped to M. thermophila genome using Bowtie2 (version 2.3.5.1). Only unique mappers were retained for downstream analyses. MACS2 (version 2.2.7.1) was employed for peak calling Mapped read density was calculated over 10 bp bins and normalized by RPGC using bamCoverage from deepTools (version3.5.1), denoised with sliding window (window size is 100 nt and step is 10 nt) and visualized with the Integrative Genomics Viewer (IGV).

ATRX and Daxx ChIP were performed as previously described (Law et al., 2010 (link)). In brief, cells were fixed in PBS with 2 mM EGS (Pierce 26103) for 45 min at room temperature followed by 1% formaldehyde for 20 min. Chromatin was sonicated to <500 bp and lysates were immunoprecipitated with 10 μg ATRX H300 (Insight Biotechnology sc-15408), 10 μg Daxx M-112 (Santa Cruz Biotechnology sc-7152), or rabbit IgG control (Dako X0903). Histone ChIPs were performed according to the manufacturer’s instructions (Millipore 17-295) using either 10 μg H3.3 antibody (Millipore 09-838), 5 μg H3K9me3 antibody (Abcam ab8898), or 2 μg H3K4me3 antibody (Abcam ab8580).