title: ChIP-seq of Homo sapiens: ABCG2+ L3.6pl tumorspheres
design: ABCG2+ L3.6pl tumorspheres were dissociated into single cells using TrypLE Express enzyme (Thermo Fisher Scientific, cat. 12-604-021) prior to fixation with 36% formaldehyde solution (Sigma-Aldrich, cat. 47608) at a final concentration of 1% for 15 minutes at room temperature with agitation. Formaldehyde was then quenched with 2.5 M glycine (final concentration of 125 mM) for 5 minutes at room temperature with agitation. Cells were washed 3 times with ice-cold PBS (each for 5 minutes) then suspended in chIP lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8, 0.1% sodium deoxycholate, 0.1% SDS and 1% Triton X-100) freshly supplemented with 1x EDTA-free protease inhibitor cocktail (Roche, cat. 04693159001) and 1x phosphatase inhibitor cocktail (Roche, cat. 4906845001). 20 cycles (30 seconds on/ 30 seconds off) were used to shear chromatin at 4C using the Bioruptor Pico sonication device (Diagenode). Fragmentation of sheared chromatin was assessed on 1.5% agarose gel in 1x TAE buffer (40 mM Tris, 20 mM glacial acetic acid and 1 mM EDTA). In each chIP experiment, a total of 10 ug of sheared chromatin was incubated with each of the following antibodies: anti-PHF5A (RRID:AB_2165365, Proteintech, cat. 15554-1-AP, dilution 1:200), anti-PHF14 (RRID:AB_2879724, Proteintech, cat. 24787-1-AP, dilution 1:200) and anti-KMT2A (RRID:AB_2918266, Proteintech, cat. 29278-1-AP, dilution 1:200) for 2 hours at 4C with agitation, followed by overnight incubation with Pierce ChIP-Grade Protein A/G Plus agarose beads (Thermo Fisher Scientific, cat. 26159) at 4C with agitation. Agarose beads were then washed 3 times with chIP wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0 and 150 mM NaCl). DNA was eluted using chIP elution buffer (1% SDS and 100 mM NaHCO3) at 65C for 30 min with agitation at 1000 rpm. To reverse cross-linking and degrade RNA, DNA samples were incubated overnight at 65C with 5 M NaCl and RNAse A (Active Motif, cat. 53040) with agitation at 400 rpm. Proteinase K (Active Motif, cat. 53040) was then used to digest proteins at 60C for 1 hour with agitation at 400 rpm. chIPped DNA was purified with Qiaquick PCR purification kit (Qiagen, cat. 28104) and quantified by Qubit 2.0 fluorometer (Thermo Fisher Scientific) using Qubit 1x dsDNA HS assay kit (Thermo Fisher Scientific, cat. Q33230). Library preparation and quality control were both conducted at CD Genomics (New York, USA) along with chIP-sequencing of 2 biological replicates per each chIP experiment and input sample using an Illumina Hiseq PE150 platform.