GSM6636983: CUT&Run ; FG; Ctrl ; H3K27me3 ; Lib203; Homo sapiens; OTHER
Chromatin Immunoprecipitation (ChIP):  Cells were crosslinked with 1% formaldehyde in 1x PBS for 10 min at room temperature. The crosslinking reaction was stopped by adding 0.125 M glycine for 5 min at room temperature. Crosslinked cells were centrifuged at 600 xg for 5 min at 4℃, and supernatant was removed. The cell pellet was washed with ice-cold 1x PBS and centrifuge at 600 xg for 5 min at 4℃. Repeat the wash step (total twice). The cell pellet was frozen by dry ice and stored at -80℃.   Crosslinked human cells were resuspended with Lysis Buffer1 (10 mM Tris-HCl [pH. 8.0], 10 mM NaCl, 0.5% NP-40, and 1x Complete Protease Inhibitor EDTA free) and incubated for 10 min on ice. 25% crosslinked Drosophila S2 cells were reconstituted with Lysis Buffer1as a spike-in control and was mixed with the human cells. Cells were centrifuged at 665 xg for 5 min at 4℃, and supernatant was removed. Cell pellet was resuspended with Lysis Buffer2 (50 mM Tris-HCl [pH. 8.0], 10 mM EDTA, 0.32% SDS, 1x Complete Protease Inhibitor EDTA free) and incubated for 10 min on ice. Cell lysate was diluted with IP Dilution Buffer (20 mM Tris-HCl [pH. 8.0], 150 mM NaCl, 2 mM EDTA, 1% TritonX-100, 1x Complete Protease Inhibitor EDTA free) and transferred into a milliTUBE ATA Fiber (Covaris). Cell lysate was sonicated by Covaris S220 (Covaris) for 3.5 min. The insoluble debris was removed by centrifuge at 12000 xg for 5 min at 4℃. Sonicated chromatin (supernatant) was transferred to a new tube and stored at -80℃.  50 to 86  uL Dynabeads Protein-G beads (10004D, Thermo Fisher Scientific) were washed twice with PBS-T (0.02% Tween-20 in 1x PBS). Desired amounts of antibodies (listed below) were conjugated to washed beads for 2 to 6 hours at 4℃ with rotation. Meanwhile, Frozen chromatin was thawed on ice. Desired amount of chromatin (listed below; determined by DNA amount) was diluted with IP dilution buffer in 4:1 ratio. Antibody conjugated Dynabeads were washed twice with IP Dilution Buffer (without Complete protease inhibitor EDTA free). Washed Dynabeads were resuspend with diluted chromatin and incubated for overnight at 4℃ with rotation. Next day, ChIPed beads were washed four times with following washing buffers: FA Lysis Buffer (50 mM HEPES-KOH [pH. 7.5], 150 mM NaCl, 2 mM EDTA, 1% TritonX-100, 0.1% Sodium deoxycholate, 1x Complete protease inhibitor EDTA free); NaCl Buffer (50 mM HEPES-KOH [pH. 7.5], 500 mM NaCl, 2 mM EDTA, 1% TritonX-100, 0.1% Sodium deoxycholate); LiCl Buffer (100 mM Tris-HCl [pH. 8.0], 500 mM LiCl, 1% NP-40, 1% Sodium deoxycholate); and 10 mM Tris-HCl [pH. 8.0]. Chromatin was eluted with TES (50 mM Tris-HCl [pH. 8.0], 10 mM EDTA, 1% SDS) for 15 min at 65℃ while shaking at 1000 rpm in ThermoMixer F1.5 (Eppendorf). The ChIP and Input chromatin were reverse crosslinked by adding NaCl (Final concentration of 200 mM) for 2 to 8 hours at 65℃. The reverse crosslinked chromatin samples were treated with 50 ng/ul RNaseA (EN0531, ThermoFisher Scientific) for 0.5 to 1 hour at 37℃. Samples were treated with 0.2 mg/ml protease K (03115828001, Roche) for 2 to 4 hours at 37℃. Finally, DNA was purified by phenol-chloroform extraction followed by ethanol precipitation. The DNA concentration was measured by Quantus fluorometer (Promega).  CUT&RUN:  20 uL of ConcanabalinA coated beads (BP531, Bangs Laboratories) were washed twice with Binding Buffer (20 mM HEPES-KOH [pH. 7.9], 10 mM KCl, 1 mM CaCl2 and 1mM MnCl2). Dissociated live cells were resuspended with Wash Buffer (20 mM HEPES-NaOH [pH. 7.5], 150 mM NaCl, 0.5 mM Spermidine and 1x Complete Protease Inhibitor EDTA free) and mixed with washed beads. The mixture was rotated for 10-15 min at room temperature. We then resuspend cell-bound beads with 100 ul (for histone mark CUT&RUN) and 50 ul (for TF CUT&RUN) Antibody Buffer (2 mM EDTA in Digitonin Buffer [0.02% Digitonin in Wash Buffer]). Desired amounts of antibodies (listed below) were added to cell-beads suspension and incubate overnight at 4℃ with rotation. Next day, beads were washed twice with Digitonin Buffer. Washed beads were treated with 700 ng/ml pA-MNase in Digitonin Buffer for 1 hour at room temperature with rotation. Beads were washed twice with Digitonin Buffer. Washed beads were resuspended with 100 ul of Digitonin Buffer and place on the cool block (BP361036, Fisher Scientific) for >10 min to cool down to 0℃. We then performed pA-MNase digestion by adding 2 ul of ice-cold 0.1 M CaCl2 and incubating for 30 min on the cool block. To stop pA-MNase reaction, 100 ul of 2x Stop Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% Digitonin, 0.05 mg/ml RNaseA and 0.05 mg/ml Glycogen) was added. To release CUT&RUN fragment, samples were incubated at 37℃ for 30 min with shaking at 300 rpm in ThermoMixer F1.5 (Eppendorf). The beads were removed by centrifuge at 16000 xg for 5 min at 4℃ and placing on a magnetic stand. Supernatant was transferred and treated with 0.1% SDS and 0.25 mg/ml ProteinaseK for 1 hour at 37℃. Finally, DNA was purified by phenol-chloroform extraction followed by ethanol precipitation. The DNA concentration was measured by Quantus fluorometer (Promega). We prepared multiplexed libraries of ChIP, Input for ChIP, and CUT&RUN samples from two replicate (two independent CRISPRi clones) using NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, NEB). The manufacture's protocol was employed with the following changes. After the adaptor ligation, we performed cleanup by AMPure XP Beads (A63880, Beckman Coulter) or MagBind Magnetic Beads (M1378-01, Omega) without size selection. After the RCR enrichment of adaptor-ligated library, we performed two rounds of size selection by the magnetic beads.