Minelute column

Cells were pre-treated with JQ1 (1 µM) or A-241 (250 nM) for 1 h prior to the addition of recombinant murine IFNy (1 ng/mL), or vehicle control, for an additional 2 h (3 h total incubation with small molecules). Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq) was performed using an improved protocol to reduce mitochondria from the transposition reaction [40 (link)]]. Briefly, 5e5 MM1.S cells were cultured in duplicate with JQ1, A-485, or DMSO vehicle as described above. Cells were washed once in ice-cold PBS and lysed in ATAC lysis buffer (0.1% Tween-20, 0.1% NP-40, 3 mM MgCl₂, 10 mM NaCl, 10 mM Tris HCl pH 7.4). Tagmentation was then performed with Tn5 transposase and 2 × TD Buffer (Nextera DNA Library Prep Kit, Illumina) for 30 min at 37 °C (in a thermocycler). Tagmented DNA was immediately purified using a MinElute column (Qiagen, #28004) and then amplified for 12 cycles using 2 × KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602) and Illumina-compatible/barcoded primers. The amplified libraries were purified using MinElute columns (Qiagen) and sequenced on an Illumina NextSeq 500 with 75 b.p. single-end reads. Library QC and quantification were performed using D1000 high-sensitivity screen tape with 4200 TapeStation Instrument (Agilent Technologies), and the size was selected between 200 and 500 bp using a Pippin Prep system (Sage Science).

To generate the library, the products of the (h)MeDIP, as well as the supernatant input DNA, were purified using Qiagen MinElute columns and eluted in 16 μl of elution buffer (Qiagen). Fourteen cycles of PCR were performed on 5 μl of the immunoprecipitated DNA using the single-end Illumina PCR primers. The resulting reactions were purified with Qiagen MinElute columns; then, a final size selection (~300–600 bp) was performed using AMPure XP beads. Quality control of the libraries was performed by an Agilent 2100 Bioanalyzer using an Agilent DNA 1000 Chip Kit. An aliquot of each library was diluted in the elution buffer to 5 ng/μl, and 1 μl was used in real-time PCR reactions to confirm enrichment of the (hydroxy)methylated region.

The ATAC-seq library was created followed previously established procedures with some minor adjustments⁵⁴ . To start, 100,000 macrophage cells were subjected to lysis using 100 μl of Lysis buffer (containing 10 mM Tris-Cl at pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, and 0.1% Tween-20). Following a centrifugation at 500g for 10 minutes at 4°C, the resulting nuclei were reconstituted with 50 μl of a transposition mixture (FC-121–1030, Illumina; comprising 1X Tagment DNA buffer, Tn5 Transposase, and nuclease-free H2O), and this mixture was then incubated for 30 minutes at 37°C in a thermomixer. The transposed DNA was subsequently purified using MinElute columns (28004, QIAGEN) and then amplified using Nextera sequencing primers and the NEB high-fidelity 2X PCR master mix for 11 cycles (M0541, New England Biolabs). PCR-amplified DNA was purified using MinElute columns (28004, QIAGEN) and ATAC-seq samples were pooled and sequenced on NextSeq 2000 P2 using paired-end sequencing.