Microchip diapure columns

WT CT215 β-cells were treated with Tm (0.2 μg/mL) for 16 h, followed by cross-linking with 1% (w/v) formaldehyde for 10 min and sonication 5 times for 30 s each at 310 W in an ice water bath with Bioruptor (Sonicbio, Samukawa, Japan). Samples were prepared by iDeal ChIP-qPCR kit (Diagenode, Seraing, Belgium) with anti-ATF4 antibody (Cell Signaling Technology, Danvers, MA, USA) or control normal rabbit IgG (Cell Signaling Technology) according to the manufacturer's instructions. Immunoprecipitated DNA was purified through MicroChIP DiaPure columns (Diagenode) and analyzed by qPCR, as described above.

Chromatin immunoprecipitation was performed using an AUTO True MicroChIP KIT (Diagenode) according to the manufacturer's protocol on an SX-8G IP-Star^® Compact Automated System (Diagenode). ChIP used 200 μL of sonicated chromatin and 3 μg of antibodies: anti-H3K27me3 (#C15410069, Diagenode), anti-EZH2 (#C15410039, Diagenode), anti-JMJD3 (#ab85392, Abcam) and anti-IgG for negative control (#C15410206, Diagenode). Antibody coating reaction with protein A-coated magnetic beads lasted 3 h, and the immunoprecipitation reaction 13 h at 4° C. Reverse cross-linking was carried out for 4 h at 65° C.
Immunoprecipitated DNA (IP) and total DNA (input) were purified by MicroChIP DiaPure columns (#C03040001, Diagenode) according to the manufacturer's instructions, and analyzed by real-time PCR.

Cells were cross-linked directly in the plates with 1% formaldehyde for 5 mn, before quenching for 5 mn in 0.125 M glycine. After PBS rinsing, cells were pelleted and snap-frozen. Chromatin preparation and immune-precipitation was done using the True MicroChIP kit (Diagenode). Briefly, 100,000 cells resuspended in lysis buffer were sonicated in a Bioruptor (Diagenode), with 3 cycles of 30 s ON, 30s OFF. Fragmented chromatin was then incubated overnight with 1 µg of H3K27me3 or H3K9me3 antibody and recovered using magnetic beads according to the manufacturer’s protocol. After reverse-cross-linking for 4 hours at 65 °C, precipitated DNA as well as input were purified using MicroChIP DiaPure columns (Diagenode), diluted 100 times before being used as a template for q-PCR. Immunoprecipitation with 1 µg rabbit IgG was used as negative control and this never yields enrichment above 0.1%. Three to four independent replicates were used.

Wild-type embryos at E12.5 were collected, dissected for trunk and limb buds, removing head and internal organs. The ChIP protocol was performed as previously described (Harada et al., 2018 ) with the following modifications. Embryonic tissues were fixed with 0.5% formaldehyde in PBS 8 min at room temperature and mechanically disrupted with a 25G syringe. The fixation reaction was stopped with 1.25M Glycine. The tissue pellet was resuspended in ChIP buffer (Harada et al., 2018 ) and incubated for 15 min on ice. Chromatin digestion was performed with Micrococcal nuclease (Cell Signaling, #10011) for 40 min at 37°C. The supernatant containing 20 μg of DNA was incubated with 20 μL of magnetic beads (Thermo Fisher Scientific) and 5 μg of PAX3 antibody, developed by C.P. Ordahl and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology Iowa City, IA 52242. After washing the beads, DNA was eluted and reverse cross-linked with 0.5 M NaCl for 4 h at 65°C and 2% proteinase K for 1 h at 50°C. DNA was purified using MicroChIP Diapure columns (Diagenode). Analyses were performed by RT-qPCR and the results expressed as a percentage of the input.

ChIP assay was carried out as previously described in ref. ^{39 (link)}, with some modifications. Briefly, we crosslinked control and UVC-treated primary hKCs, lysed cells, and sonicated lysates with the Bioruptor Pico device (Diagenode) at 8 °C for 30 cycles (30 s on, 30 s off). We quantified chromatin with the Qubit system (Thermo Fisher) and incubated 2 ug of crosslinked chromatin with 5 ug of normal rabbit IgG (SCBT, sc-2027) or anti-p53 antibody (rabbit polyclonal, Diagenode C15410083). We used magnetic protein G beads (Dynabeads, Invitrogen 10003D) for immunoprecipitations. Inputs correspond to 1% of the total DNA sample. We de-crosslinked the inputs and immunoprecipitates and purified DNA with MicroChIP DiaPure columns (Diagenode C03040001). We analyzed TINCR p53 RE enrichment over the input chromatin by quantitative real-time PCR (Applied Biosystems) using FastStart Universal SYBR Green (ROCHE) with the following primers: P53 RE TINCR int1 Fw 5′-CAACATGGTGAAACCCCATC-3′, and P53 RE TINCR int1 Rv 5′-CGCCTCCCAGACTCAAG-3′.

ChIP–seq on L3 EDs were performed as described previously^{41 (link)}, with minor modifications, and 400 EDs were used per replicate. If necessary, several dissection and/or collection batches were frozen in liquid nitrogen and stored at −80 °C to collect sufficient material. Chromatin was sonicated using a Bioruptor Pico (Diagenode) for 10 min (30 s on, 30 s off). PH antibodies^{67 (link)} were diluted 1:100 for immunoprecipitation. After decrosslinking, DNA was purified using MicroChIP DiaPure columns from Diagenode. DNA libraries for sequencing were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Sequencing (paired-end sequencing 150 bp, roughly 4 Gb per sample) was performed by Novogene (https://en.novogene.com/). All experiments were performed in biological duplicates.