Bioruptor machine

ChIP assays were performed using the ChIP kit (Millipore) with the usage of anti-FOXO1 antibody (1:50; ab39670, Abcam) as described^{67 (link)}. Briefly, cells were fixed with 1% formaldehyde and lysed with lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS, 0.2 mM phenylmethane sulfonyl fluoride, 1 μg ml⁻¹ aprotinin, 2.5 μg ml⁻¹ leupeptin, 1 μg m⁻¹ pepstatin). The cell lysates were sonicated with a Bioruptor machine (Diagenode) for 12–18 min with cycles of 30 s pulses and 1 min pauses to shear the DNA to a final size of 200–500 bp. After preclearing with protein A beads (Repligen) for 1–2 h, the antibody was added and incubated overnight at 4 °C. Protein A-agarose beads were added and incubated for 1–2 h. The complex was washed with low-salt buffer, high-salt buffer, and LiCl buffer and twice with Tris-EDTA buffer, followed by elution in a buffer containing 1% SDS and 0.1 M NaHCO₃. The crosslinks were reversed, and the DNA was purified with a QIAquick PCR purification kit (Qiagen) and subjected to analysis by real-time qPCR. Chromatin immunoprecipitates for proteins were amplified by qPCR, normalized to input, and calculated as percentage of input. The PCR primers for ChIP assays are listed in Supplementary Table 4.

DNA was fragmented to 550–650 bp using a BioRuptor machine (Diagenode, Belgium). Samples were prepared following the TruSeq Nano DNA Sample Preparation Guide (Part#15041110Rev.D). Sequencing was performed using an Illumina NextSeq 500 sequencer with NextSeq Mid Output v2 Kit chemicals (Illumina Inc., San Diego, CA 92122, USA). Read- and assembly-based analyses were performed using the METAnnotatorX bioinformatic platform described below in this manuscript. Mapping of reads on nucleotide sequences was performed using the software BowTie2 [19 (link)] and retrieval of mapping or non-mapping reads was performed using the Sequence Alignment/Map tools (SAMtools) 43 [20 (link)].