GSM5505998: Parental EW8 cell line - FLI1 ChIP-seq; Homo sapiens; ChIP-Seq
Prior to immunoprecipitation (IP), antibodies targeting proteins of interest were conjugated to magnetic beads as follows. For each IP, 100 µl of magnetic Protein A Dynabeads (Thermo Fisher Scientific, 10002D) were washed three times in 1 ml BSA blocking solution (0.5% w/v sterile-filtered Bovine Serum Albumin in H2O) and resuspended in 250 µl of the solution. Beads were then rotated overnight at 4ºC with antibody, using 5 µg of antibody to detect H3K27ac (Abcam, # 4729) or 10 µg of antibody to detect TFs (anti-HA, Abcam, # ab9110; anti-FLI1, Abcam, ab15289). For comparative studies (i.e., comparing the relative binding of EWS/FLI), 2 µg of spike-in antibody recognizing a Drosophila-specific histone variant was added (Active Motif, # 61686). The following morning, the antibody-conjugated beads were washed four times in 1 ml BSA blocking solution and then resuspended in 100 µl of the solution and stored at 4ºC until use. To prepare sheared chromatin, Ewing sarcoma cells (20 million cells per ChIP reaction) were collected in a 1.5 ml reaction tube and washed twice in 1 ml PBS. Cells were then crosslinked by resuspension in 1 ml PBS containing 1% methanol-free formaldehyde (Thermo Fisher Scientific, # 28906) and rotated for 10 minutes at room temperature at 12 RPM. The fixation reaction was quenched by adding 100 µl of 1.25M glycine (Sigma Aldrich, # G7126) and 100 µl 1M Tris-HCl pH 8.0 (Thermo Fisher Scientific, # 15568025). Cell pellets were washed twice with 1 ml PBS at room temperature and resuspended in 1 ml of SDS Lysis Buffer (0.5% SDS, 5 mM EDTA, 50 mM Tris-HCl pH 8.0) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, # PI78429) and incubated at room temperature for 2 minutes with gentle agitation. Lysates were centrifuged at 15,000xg for 10 minutes at 4°C. The supernatant was discarded and the transparent nuclear pellet was re-suspended in 950 µl of ChIP IP Buffer (2 parts SDS Lysis Buffer and 1 part Triton Dilution Buffer, which was composed of 100 mM Tris-HCl pH 8.0, 100mM NaCl, 5mM EDTA, 0.2% NaN3, and 5% Triton X-100) supplemented with protease inhibitor cocktail and transferred to a milliTUBE (Covaris Inc, # 520130) for shearing of chromatin. Sonication was performed on an E220 Focus Ultra Sonicator (Covaris) using the settings 5% duty cycle, 140W peak power, 200 cycles per burst, at 4°C, at 30 minutes per milliTUBE. Following sonication, sheared chromatin was transferred to a 1.5 ml reaction tube and centrifuged at 15,000xg for 10 minutes at 4°C to pellet debris. The supernatant, constituting cleared sheared chromatin, was transferred to a new reaction tube. To prepare the ChIP DNA Input sample, 5 µl of sheared chromatin was transferred to a PCR strip-tube and mixed with 40 µl de-crosslinking buffer (100 mM NaHCO3 and 1% SDS buffer), 1 µl RNAse A (Thermo Fisher Scientific, # 12091021), and 1 µl Proteinase K (Thermo Fisher Scientific, # AM2546). The tube was incubated for 2 hours at 65°C in a thermal cycler to de-crosslink DNA-protein covalent bonds. DNA was then isolated using Agencourt AMPure XP bead-based purification at a 1.2X ratio (Beckman Coulter, # A63881), eluted in 50 µl H2O, and stored at -20ºC. The remaining sheared chromatin was divided or pooled according to the target of interest; at least 5 million cells were used for immunoprecipitation of histone marks and 40 million cells for transcription factors (TFs). Each IP reaction was brought up to a total volume of at least 1 ml with ChIP IP buffer in a 1.5 ml tube. Pooled reactions were conducted in 2 ml or 5 ml reaction tubes. 50 ng of Drosophila spike-in chromatin was added for each H3K27ac ChIP reaction, while 20 ng of spike-in chromatin was added for each TF ChIP reaction. The 100 µl conjugated bead-antibody solution was then added to the sheared chromatin. IP reactions were rotated overnight at 4°C. The following day, ChIP reactions were washed twice in 1 ml low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), then twice in 1 ml high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then twice in 1 ml lithium chloride buffer (0.25 M LiCl, 1% IGEPAL-CH 630, 1% sodium deoxycholate, 10 mM Tris-HCl pH 8.0, 1 mM EDTA), and then once in 700 µl Tris-EDTA buffer (Sigma Aldrich, # 93283). All washes were performed on ice using buffers that had been stored at 4ºC. Chromatin was eluted from beads using 100 µl fresh ChIP Elution Buffer (1% SDS, 0.1M NaHCO3) and rotated at room temperature for 15 minutes. Eluate was transferred to PCR tubes and mixed with 8 µl 2.5M NaCl, 1 µl RNAse A, and 1 µl Proteinase K. Samples were de-crosslinked for 12-16 hours at 65°C on a thermal cycler. ChIP DNA was extracted from the de-crosslinked samples using AMPure XP beads at a 1.2X ratio and eluted in 20 µl of H2O. DNA from input and ChIP samples were quantified using a Qubit fluorometer (Thermo Fisher) and the Qubit dsDNA high sensitivity assay (# Q32851). ChIP-seq libraries were prepared using the SMARTer ThruPLEX DNA-Seq kit (Takara Bio, # R400674). H3K27ac ChIP-seq samples were amplified 4 PCR cycles. TF ChIP-Seq samples were amplified 10 PCR cycles. Samples were single-indexed (Takara Bio, # R400695). The concentration of DNA in each library was measured by Qubit and the average of DNA fragment sizes was measured by Tapestation. The approximate molarity of each library was calculated. Each library was diluted to a concentration of 4 nanomolar and equal volumes of each library were pooled. The approximate molarity of the pool was then determined by Qubit and Tapestation and adjusted for sequencing according to Illumina standards. Libraries were sequenced, in 37-basepair paired-end fashion for 75 cycles (Illumina Inc, # 20024906), at a depth of roughly 30 million reads per sample, on the Illumina NextSeq 500 instrument.