H3k27ac antibody

H3K27Ac ChIP in 22RV1 cells was performed as previously described (Pomerantz et al., 2015 (link)). Ten million cells were fixed using 1% formaldehyde (Thermo fisher, Waltham, MA) for 10 minutes at 37°C. Chromatin was sheared in ice cold lysis buffer (50mM Tris, 10mM EDTA, 1% SDS with protease inhibitor) to 300–500 base pairs using the Covaris E210 sonicator. The sample was incubated with 1 µg H3K27Ac antibody (Diagenode, C15410196, Denville, NJ) coupled with protein A and protein G beads (Life Technologies, Carlsbad, CA) at 4°C overnight. The chromatin was washed with RIPA washing buffer (0.05M HEPES pH 7.6, 1 mM EDTA, 0.7% Na Deoxycholate, 1% NP-40, 0.5M LiCl). After decrosslinking, IP DNA as well as its input were extracted using Qiagen Qiaquick columns, and sequencing libraries prepared using the ThruPLEX-FD Prep Kit (Rubicon Genomics, Ann Arbor, MI). Libraries were sequenced using 75-base pair single reads on the Illumina platform at the Dana-Farber Cancer Institute.

ChIP experiments were conducted as described previously (Wang et al., 2009 (link)) and were done in triplicates. Chromatin from approximately 1 × 10⁷ fixed cells was sonicated to a size range of 200-300 bp. Solubilized chromatin was subjected to immunoprecipitation with the ER antibody, Ab10 (Lab Vision corporation) and SC-543 (Santa cruz), HA antibody (Ab9110, Abcam), H3K27Ac antibody (C15410196, Diagenode) or FOXA1 (ab5089,Abcam) bound to protein A and protein G beads (Life Technologies). A fraction of the sample was not exposed to antibody to be used as control (input). The samples were reversed crosslinked, treated with proteinase K, and DNA was extracted. DNA sequencing libraries were prepared using the ThruPLEX-FD Prep Kit (Rubicon Genomics). Libraries were sequenced using 50 bp reads on the Illumina Nextseq500 at the Dana-Farber Cancer Institute.

FiTAc-seq (68 (link)) was used to profile H3K27ac ChIP-seq on FFPE samples. Sample selection was performed with dual-IHC staining assessing tumor enrichment with SOX10 antibody and MHC-I expression status. Accordingly, areas of interest were marked and samples were classified into low or high MHC-I categories. The selected areas of interest were manually macrodissected from 10 unstained sections (10 μm each) per case. Briefly, the macrodissected tissues were washed with xylenes to remove paraffin, rehydrated in an ethanol/water series and incubated overnight with 200 μl of lysis buffer (1%SDS, 50 mM Tris-HCl pH8, 10 mM EDTA) on a thermomixer set at 65C, and shaking at 1000rpm. Next, samples were put on ice, supplemented with protease and deacetylase inhibitors and sonicated with Covaris E220 instrument for 5 min (PIP105, 5%DF, 200cbp) in 1mL AFA Fiber milliTUBEs. Solubilized chromatin (ranging from 450 ng to 3 μg) was pre-cleared, diluted, and immunoprecipitated overnight with 3 μg of H3K27ac antibody (Diagenode, cat#A1723–0041D, lot#C15410196) following a low-input protocol; and finally washed, eluted and DNA purified. Libraries were prepared with Accel-NGS 2S Plus DNA Library Kit (Swift Biosciences, cat. no. 21024) on an automated NGS workstation, amplified for 14 cycles, and sequenced on a Next-seq instrument (PE35).