Bioruptor pico system

ChIP experiments were performed using the Ideal-ChIP for transcription factor kit (Diagenode, Liège, Belgium) following the manufacturer’s protocol. Specifically, 4 × 10⁶ BZ cells per well of 6-well plates grown in 1% DCC medium were treated by vehicle (ethanol), Dex 10^-7 M or Dex 10^-7 M together with RU 10^-6 M for 1 h. Cells were then fixed by a mix of formaldehyde 16% and fixation buffer from the kit (4: 1 ratio), 1/100 volume final for 8 min. ChiP experiments were performed according to Le Billan et al [52 ]. Chromatin samples (300 μl) were sheared by 12 cycles 30 s ON and 30 s OFF with the Diagenode Bioruptor Pico system and 2.5 μl samples were kept for input measurements. Two hundred and fifty μl of sheared chromatin samples were incubated overnight at 4 °C with 1 μg IgG from the kit as control or 5 μg anti-GR antibody H300 (Santa Cruz sc-8992-X [53 (link)]). Chromatin shearing was controlled on a 1.5% agarose gel and typically a smear was visualized ranging from 100 to 500 bp. qPCR amplifications of eluted DNA were performed with primers encompassing a short upstream sequence of exon IV, or on Per1 and Ucp1 promoters (Additional file 1: Table S1 for primer sequences). Raw data are expressed as percentage of inputs, according to the Percent of Input Method, ChIP analysis; Thermo Fischer Scientific.

Two milliliters of blood were drawn from the wing vein of each chicken in a centrifuge tube containing anticoagulant (EDTA-2 K) and stored at − 80 °C until use. Genomic DNA (10 μg) in each blood sample was extracted using a DNA extraction kit (DP326, TIANGEN Biotech, Beijing, China) and fragmented using a Bioruptor Pico System (Diagenode, Belgium). DNA fragments around 350 bp were selected using SPRI beads (Beckman Coulter, IN, USA). DNA-sequencing libraries were prepared using Illumina TruSeq® DNA Library Prep Kits (Illumina, CA, USA) following the vendor’s instructions. The libraries were subject to 150 cycles paired-end sequencing on an Illumina Novaseq 6000 platform (Illumina, CA, USA) at ~30X coverage.