title: GSM4551742: NFYA_CUTRUN_cloneB8p; Homo sapiens; OTHER
library_construction_protocol: Briefly, 1 million cells were collected and frozen in 10% DMSO in FBS, as necessary. Frozen cells were thawed, pelleted, and washed once with PBS, and  processed according to the following steps: 1) addition of 0.5 mL of NE buffer (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 0.5 mM Spermidine, 0.1% Triton X-100, 20% Glycerol and 1x protease inhibitor cocktails from Sigma) for nuclei isolation. Nuclei were pelleted at 600 x g for 3 min, resuspended with 0.4 mL of NE buffer. 2) 25 uL BioMagPlus Concanavalin A beads slurry was washed twice and resuspended in 200uL Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2), and   Binding Buffer is added to the nuclei suspension. The mixture was thoroughly mixed on a rocker for 10 min to allow binding to nuclei to the beads. 3) The nuclei were pelleted with a magnet stand and blocked with Wash Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA and 1x protease inhibitor cocktails from Sigma) supplemented with 2 mM EDTA. After washing once with Wash Buffer, nuclei were resuspended in 200 uL Wash buffer containing 2 ug of antibody, and incubated overnight to allow antibody binding. 4) The next day, wash the nuclei twice on a magnet stand with wash buffer and incubate with 1:1000 pA-MN in 200 uL Wash Buffer. After 1 hr, wash twice to remove unbound pA-MN. 5) Resuspend the nuclei in 150 uL Wash Buffer, chill on a 0°C metal block that sits in water-ice mixture. Add 3 uL 100 mM CaCl2 to activate pA-MN, and incubate at 0°C for 60 min. 6) The reaction was stopped by adding 150 uL 2XSTOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 mg/mL RNase A and 40 mg/mL glycogen). The protein-DNA complex was released by centrifugation and then digested by proteinase K at 50°C overnight. DNA was extracted by ethanol precipitation, followed by Qubit fluorometer and bioanalyzer quality control. Library preparation of CUT&RUN was performed using NEB Ultra II library preparation kit with important modifications. Briefly, less than 30 ng DNA is used as input. End prep was performed at 20°C for 30 min and then 50°C for 60min (instead of 65°C for 30 min in the manual). The reduced temperature prevents melting of short DNA fragments. 5 pmol adaptor was added and ligated to end prep products at 20°C for 15 min. USER enzyme was then used to cleave the uracil in the loop. 1.7x volume of AMPure beads were used to purify the ligation product. The use of 1.7x beads is essential for recovery of short fragments. To amplify the library, the ligation product was mixed with 2x Ultra II Q5 mix, universal primer and index primers. PCR was carried out as follows: 98°C 30 s, 12 cycles of 98°C 10s and 65°C 10s, and final extension 65°C 5 min. The PCR product was subjected twice to double size selection  with 0.8x and then 1.2x AMPure beads. The purified library was quantified with Qubit and Tapestation. Libraries were pooled at similar molar amount and sequenced using Nextseq500 platform. Paired-end sequencing, read length 42 bp x 2, 6 bp index was performed. For detailed protocol please see: dx.doi.org/10.17504/protocols.io.wvgfe3w
attributes.tag: GEO Accession
attributes.value: GSM4551742