GSM4551311: NFYA_ChIP_BCLKOHDP2_shNFYA; Homo sapiens; ChIP-Seq
Five million differentiated HUDEP cells were collected and fixed with1% formaldehyde for 5 minutes at room temperature, and quenched with 125 mM glycine. Cells were washed with ice-cold PBS twice and resuspended in 0.13 mL lysis buffer (50 mM Tris-HCl 8.0, 10 mM EDTA, 0.5% SDS) and sonicated in a microtube (Covaris, 520045) with Covaris E220 ultrasonicator (Covaris) to an average of 300-500 bp. Sonicated chromatin was diluted with 1 mL ChIP dilution buffer (20 mM Tris-HCl 8.0, 2 mM EDTA, 1% Triton X-100, 300 mM NaCl, protease inhibitor), followed by adding 20 uL Dynabeads protein G (Thermo Fisher Scientific) and 3 ug antibody (NFYA, ab139402, Abcam). The mixture was incubated on a rotator overnight and washed with the following buffers: twice with RIPA150 (20 mM Tris-HCl 8.0, 1 mM EDTA, 1% Triton X-100,150 mM NaCl, 0.1% Sodium Deoxycholate, 0.1% SDS), twice with RIPA500 (20 mM Tris-HCl 8.0, 1 mM EDTA, 1% Triton X-100, 500 mM NaCl, 0.1% Sodium Deoxycholate, 0.1% SDS), twice with LiCl buffer (10 mM Tris-HCl 8.0, 1 mM EDTA, 0.5% NP-40, 250 mM LiCl, 0.5% Sodium Deoxycholate), twice with TE buffer (10 mM Tris-HCl 8.0, 1 mM EDTA). The washed chromatin was eluted using 300 uL Elution buffer (50 mM Tris-HCl 8.0, 10 mM EDTA, 1% SDS, 150 mM NaCl, 0.1 mg/mL Proteinase K) and decrosslinked at 65°C overnight. DNA was extracted with phenol-chloroform, and precipitated by adding 2.5x volume of absolute ethanol followed by centrifuging at 14,000 rpm for 15 minutes. The pellet was washed once with 75% ethanol, then dried and dissolved with 50 uL TE buffer. Library was prepared with NEBNext Ultra II DNA Library Prep Kit (NEB) according to manufacturer's protocol.
GEO Accession
GSM4551311