Micrococcal nuclease

ChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, #9003) according to manufacturer’s recommendations. Following treatment, MDA-MB-231 cells were cross-linked with 37% formaldehyde (Sigma Aldrich, F8775) at a final concentration of 1% for 10 min at room temperature. Chromatin was digested by adding 1 µL of micrococcal nuclease (Cell Signaling Technology, #10011) per IP prep and incubation for 20 min at 37 °C. Samples were then subjected to sonication. ChIP was performed using anti-XBP1s (Cell Signaling Technology, #40435, 1:50) or normal rabbit IgG (Cell Signaling Technology, #2729) antibody. Immunoprecipitated DNA fragments were purified and analyzed by qPCR using primers designed against the promoter of PERK or exon 1 of DNAJB9 (Cell Signaling Technology, #79879). Results were calculated using the percent input method. The acquired ΔCt values were used to assess statistical significance between treatments.

ChIP was performed as described by Attaai et al. (2018) (link). The SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technology, #9003) was used according to the manufacturer’s instructions. Moreover, 1 × 10⁶ BV2 cells/10 cm cell culture dish were treated with TGFβ1 (5 ng/ml) for 2 h, and proteins were cross-linked with 1% formaldehyde for 10 min. Chromatin digestion was performed with 0.25 μl Micrococcal Nuclease (#10011, Cell Signaling Technology) for 20 min at 37°C. The nuclei were lysed with three sets of 20-s pulses using a Bioruptor sonicator (Diagenode, Liège, Belgium). Normal rabbit IgG (#2729, Cell Signaling Technology) as a negative control, anti-Histone H3 (#4620, Cell Signaling Technology) as a positive control as well as anti-Smad2 (#5339, Cell Signaling Technology) and anti-Smad4 (#38454, Cell Signaling Technology) were used for immunoprecipitation. Chromatin was incubated overnight at 4°C and, after incubating with protein G magnetic beads for 2 h and washing in magnetic separation racks (Cell Signaling, #7017), elution of chromatin was achieved. Cross-linking was reversed using 2 μl Proteinase K (Cell Signaling, #10012) for 2 h at 65°C. Finally, DNA purification in spin columns was performed, and promoter fragments were amplified using qPCR. Data are expressed as 2^–ΔCT for the SBE of interest normalized to the rabbit IgG isotype control.

Snap-frozen lumbar DRG were gently thawed on ice and incubated with 18.5% formaldehyde for protein–DNA crosslinking. After 10 min, glycine was added to a final concentration of 0.125 M. The DRG were mechanically dissociated and centrifuged (800 g, 5 min, 4 °C). The nuclear pellet was incubated with micrococcal nuclease (Cell Signaling) in MN buffer (10 mM TrisCl pH=7.5, 4 mM MgCl₂, 1 mM CaCl₂, PIC) at 37 °C for 10 min. The samples were centrifuged (10,000 g, 1 min, 4 °C) and chromatin-containing pellet was sonicated (20 s, 3 pulses). The samples were precleared using protein A/G magnetic beads. Then, 60 µg chromatin from each sample was incubated overnight at 4 °C with 1 μg of an appropriate NF-kB subunit antibody (Supplementary Table T12) along with magnetic protein A/G beads. The rest of the procedure was carried out using ChIP Magna kit as per manufacturer’s instructions (EMD, Millipore). The quantification of Pomc promoter fragments specific for NF-kB binding sites were quantified using specific primers (Supplementary Table T1) using a quantitative real-time PCR.
For ChIP analysis on AtT-20 cells, cells were exposed to either 5 mM or 20 mM glucose-containing medium with reduced serum. Post 12 h of exposure, the cells were cross-linked with formaldehyde to a final concentration of 1%. The rest of the procedure was same as above.

1×10⁸ HH514-16 cells were induced with 3mM NaB with or without phosphonoacetic acid (PAA) (200 μg/ml) for 36 hours prior to performing iPOND as described previously [29 (link)]. Briefly, cells were pulsed with 10 μM EdU for 15 min, spun down, cross-linked with 1% formaldehyde for 20 min, and quenched with 0.125 M glycine for 5 min. For click chemistry, cells were incubated with 10 μM biotin-azide in click reaction buffer for 2 hours. Nuclei were isolated and digested with 1 μL of micrococcal nuclease (10011, Cell Signaling) at 37°C for 20 min and suspended in cold lysis buffer (1% SDS, 50 mM Tris, pH 8.0), and subjected to sonication using a microtip sonicator to break nuclear membranes. After removing debris, the supernatant was incubated with 100 μL of streptavidin agarose beads (69203, EMD Millipore) overnight at 4°C and washed three times with lysis buffer and one time with 1M NaCl. Protein-DNA complexes were eluted with 2X Laemmli buffer at 95°C for 25 min and subjected to immunoblotting.

ChIP assays and qPCR were performed with Simple ChIP Enzymatic Kit (Cell Signaling Technology, Danvers, MA, USA), following the manufacturer’s instructions with minor adaptions of the number of cells for each immunoprecipitation and chromatin digestion. Briefly, 6 × 10⁷ cells either prior to incubation with trehalose (T0) or after incubation with trehalose for 48 h (T48) were used for each immunoprecipitation. To crosslink the cells, formaldehyde was added to a final concentration of 1%, and cells were incubated for 10 min at room temperature. The cells were washed with ice-cold PBS twice, followed by preparation of nuclei and chromatin digestion. To digest DNA to a length of approximately 150–300 bp, 2 μL micrococcal nuclease (Cat#10011 by Cell Signaling Technology) was added and the samples were incubated for 20 min at 37 °C. After terminating digestion by addition of EDTA, lysates were sonicated using three or four pulses of 20 s each at setting 7 on a handy sonicator (UR-21P, Tomy Seiko, Tokyo, Japan). Approximately 5 μg digested chromatin samples and 0.25 μg mouse monoclonal anti-DDDDK-tag (M185-3, MBL, Tokyo, Japan) were used for each immunoprecipitation, and the samples were incubated on a rotating platform at 4 °C overnight. After purification using a spin column, qPCR was performed using the TB Green Premix Ex Taq II (Takara Bio) with specific primers (Table S9).

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.