Ab8580

ESCs were fixed in 4% paraformaldehyde for 30 min at room temperature and then permeabilized with 0.1% Triton X-100 for 30 min. Then cells were blocked in 10% goat serum for 1 h at room temperature. Primary antibodies against Dppa3 (1:200; R&D systems, MAB2566), Zscan4 (1:1000, Millipore, AB4340), H3K4me3 (1:1000, Abcam, ab8580), γH2AX (1:1000, Cell Signaling Technology, 80312), and 5mC (1:200, Abcam, ab10805, the cells were treated with 2 M HCl for 30 min, then neutralized with Tris-HCl of pH 8.0) were diluted in 1% normal serum (v/v) and incubated overnight at 4 °C and detected with the respective secondary antibodies including Alexa Fluor 594 secondary antibody (1:500; Proteintech, SA00006-4), Alexa Fluor 647 secondary antibody (1:500; Abcam, ab150159), Alexa Fluor 555 secondary antibody (1:500; Beyotime, A0460) at room temperature for 1 h and labeled with DAPI (Sigma) to visualize the nuclei. Sample images were captured using laser scanning confocal microscopy.

CUT&Tag was performed as described earlier starting from nuclei [28 (link),29 (link)]. First, nuclei were isolated from the sorted macrophages. In short, the macrophages were centrifuged for 5 min at 4 °C, 500 rpm, supernatant was removed, and the cells were lysed on ice in 1 mL of nucleus extraction buffer (1 × prelysis buffer from the EpiGentek EpiQuick Total Histone Extraction Kit, OP-0006-100). To stop the lysis reaction, 1 mL of PBS+1%BSA was added, and nuclei were collected through centrifugation for 5 min at 4 °C, 500 rpm. The supernatant was removed, the nuclei were resuspended in PBS+1% BSA, and a sample was visually inspected for viability, purity, and abundance of nuclei under the microscope. Next, CUT&Tag libraries were created according to the published CUT&Tag protocol for nuclei [29 (link)]. All buffers were supplemented with 5 mM sodium-butyrate (Sigma, 303410) and 1X complete protease inhibitor (Merck, 1187358000). Protein lo-bind tubes (Eppendorf, EP0030108116) were used to reduce sample loss. Antibodies against H3K18la (PTM-Bio, PTM-1406), H3K4me3 (Abcam, ab8580), H3K27me3 (Cell Signaling Technology, C36B11) and H3K27ac (Abcam, ab4729) were used in this study. Libraries were indexed using Nextera Indexes, and 150-bp paired-end sequencing was performed on Illumina Novaseq instruments.

After stimulations, the cells were fixed with 1% formaldehyde, lysed, and sheared. The DNA was quantified, and 5 mg of total chromatin was immunoprecipitated with specific antibodies and Dynabeads Protein G (Novex/Life Technologies #10009D). The DNA was then reverse crosslinked, purified, and quantitated by quantitative PCR (qPCR) amplification with primers designed at the promoter sites of the IFNB (F-5′-TCGTTTGCTTTCCTTTGCTT-3′, R-5′-CCCACTTTCACTTCTCCCTTT-3′) and RSAD2 (F-5′-CCTGGCATACAGGACACCTT-3′, R-5′ AAGAGTTCTGTCCGCTTCCA- 3′) genes for RNA Polymerase II and H3K4me3 ChIP. For STAT1 ChIP, primers targeting the STAT1-binding sites for CXCL10 (F-5′-AAAGGAACAGTCTGCCCTGA-3′, R-5′-CACTGATGTCCTCCTGCTCA-3′), RSAD2 (F-5′- TTGGCCCTGTTTCAACTTTC-3′, R-5′-TCTGAGCAACCTGTCATTGG-3′), IFI16 (F-5′- ATTTCTCATCCCCCATTTCC-3′, R-5′-GAGACTCCTCCCACCAGTGT-3′), and MX2 (F-5′ AGTTTGGGGACCACTCTGTG-3′, R-5′- CTGCTCCGTCATCAACAAAC-3′) have been used. Antibodies used were against RNA Polymerase II (RNA Pol-II; Active Motif #39097), Histone H3 trimethylated at Lysine 4 (H3K4me3; Abcam # ab8580), STAT1 (Cell Signaling Technology, #9172), or control IgG isotype (Abcam # ab37415 or Cell Signaling Technology #5415) as per manufacturer’s recommended instructions. Data was calculated as the percentage fraction of total input DNA and using IgG isotype as control.