Afa technology

We sequenced three samples in the exponential phase from different experimental realizations for each temperature. In addition, we sequenced three stationary samples at three different temperatures. DNA samples were sheared by ultrasound using Covaris AFA technology. Libraries were then prepared using the PCR-free NEBNext Ultra II DNA Library Prep Kit for Illumina. Sequencing was performed on a Novaseq6000 using paired-end 150 bp reads.

The sequencing library was prepared by random fragmentation of genomic DNA, followed by 5’ and 3’ adapter ligations. Briefly, 100 ng genomic DNA was fragmented using adaptive focused acoustic (AFA) technology (Covaris Inc., Woburn, MA, USA). The fragmented DNA was end-repaired and ligated to TruSeq indexing adapters using the Illumina TruSeq DNA Nano Library Prep Kit according to the manufacturer’s instructions (Illumina Inc., San Diego, CA, USA). The resulting libraries were quantified through a qPCR-based assay using the KAPA Library Quantification Kit for Illumina Sequencing platforms according to the manufacturer’s instructions (Kapa Biosystems, Woburn, MA, USA). The libraries were qualified using an Agilent Technologies 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA).

Chromatin immunoprecipitation (ChIP) was performed using the SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling #9005). Briefly, 12 × 10⁶ cells were collected and fixed with 1.5% of fresh formaldehyde, incubated for 10 min at 20 °C, and stopped with 125 mM glycine for 5 min at 4 °C. Next, the SimpleChIP^® Plus Enzymatic Chromatin IP protocol was followed. The Adaptive Focused Acoustics™ (AFA) Technology from Covaris (Waltham, MA, USA) was used to sonicate chromatin for 4 min with 5% of sonication (75 Watts) two times. Chromatin was precleared with protein G agarose beads (Cell Signaling Technology, Waltham, MA, USA) for 2 h at 4 °C and immunoprecipitated with 8.5 μL of MEIS1-specific antibodies (Atlas Antibodies HPA056000), a negative control IgG antibody, or a positive control Histone H3 antibody (Cell Signaling Technology) overnight at 4 °C.
PCRs were performed in triplicate using the HotStarTaq Master Mix (QIAGEN), and the primer sequences used are listed in Table S5.
All immunoprecipitations were carried out in triplicate, using different chromatin preparations.