Instrument: Illumina Genome Analyzer
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: For the HPC7 cell line ChIP-seq, 500.000 cells were fixed either with 0.1% PFA (H4K16ac profile) or 1% (GATA-1 profile) for 10 minutes at room temperature and quenched with 0.125M glycine. Lysates were transferred into 0.1 mL Bioruptor Microtube (Diagenode C30010015, Liège, Belgium) and the chromatin was sheared using an NGS Bioruptor Sonicator (Diagenode) at default intensity and cycles of 30'' ON/30'' OFF Sonication time was calibrated for the different cell types: 10 minutes for MEPs and 15 minutes for HSCs/HPC7. After sonication, samples were centrifuged at high speed for 15 minutes at 4oC to pellet precipitated SDS. Chromatin extracts were diluted 1 to 5 with Sonication Equilibration Buffer (10 mM TrisCl, 140 mM NaCl, 0.1 % Sodium Deoxycholate, 1% Tx-100, 1 mM EDTA, 1X Protease Inhibitors (Roche) (Lara-Astiaso et al., 2014) . Chromatin was pre- cleared before adding the antibodies by adding 50 μL of protein A/agarose DNA bead; samples were kept in constant rotation for 2 hours at 4oC. After that, chromatin samples were centrifuged at 3000 rpm for 5 minutes at 4oC, supernatants was transferred to fresh 1.5 mL micro centrifuge tubes and 2 μg of anti-MOF (#A300-992A, Bethyl), anti-GATA-1 (#181544, Abcam) or anti-H4K16ac (#07-329, Millipore) was added to the diluted chromatin extracts and incubated for 20 hours at 4oC. Samples centrifugation supernatant was collected and 20 μL of protein A-dynabeads (Thermo Fisher Scientific), previously blocked with 1% BSA, was added to the samples, samples were kept in constant rotation for 2 hours at 4oC. Then, we centrifuged the samples at 3000 rpm at room temperature and supernatants were kept. Samples were washed with high-salt buffer (50 mM HEPES pH 7.9, 500 mM NaCl, 1mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% deoxycholate) twice, once with LiCl buffer (10 mM TE, 250mM LiCl, 0.5% NP-40, 0.5% deoxycholate), once with TE (10Mm Tris-HCl pH 8.0, 1mM EDTA) and then eluted in 10 μL of elution buffer (Qiagen). Eluate was treated with 2μl of RNaseA (Qiagen) for 30 minutes at 37oC, followed by 2.5 μl of Proteinase K (Thermo-fischer) treatment for two hours at 55oC. After that, temperature was increased to 65oC for 8 hours to revert formaldehyde crosslinking. Chromatin was quantified by Qubit Fluorometric Quantitation (Thermo Fisher Scientific) DNA high- sensitivity kit (#Q32854, Thermo Fisher Scientific) and quality was evaluated by fragment analyzer. The library was completed and amplified through a PCR following the DeepSeq using the NEBNext® UltraTM DNA Library Prep Kit for Illumina default instructions (NEB #E7645). ChIP libraries were sequenced using an Illumina HiSeq 3000. FASTA files were then transferred to Galaxy Platform (Afgan et al., 2016), where we conducted a) Genome alignment (mm10) by Bowtie2 version 2.3.0.1 (Langmead and Salzberg, 2012) using default parameters. B) Quality control was assessed by deeptools (Ramírez et al., 2016). And C) and calling Peaks by MACS2 software (Feng et al., 2012), in which bandwidth was set to 300, lower mfold bound to 5, upper mfold bound to 500. Peak detection was based on q-value (minimum FDR cutoff as 0.05). For build model we selected the shifting mode.