GSM9218576: 3367696_JWH007_Group_INPUT; Homo sapiens; ChIP-Seq
Cells were fixed with 1% formaldehyde for 5 min with gentle rocking at room temperature, and chromatin was prepared using the Covaris TruChIP Chromatin Shearing Kit (Covaris, 520154). Chromatin was sheared by the Covaris M220 ultrasonicator (duty factor of 10, cycles/burst 200 for 10 min at set point 6°C). After clarification by centrifugation at 8,000 x g for 10 mins, the chromatin was moved to a new 1.5 mL Eppendorf tube, and the buffer was amended for immunoprecipitation (final concentration: 50 mM Tris HCL pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 % SDS, 0.5 % Na Deoxycholate plus protease inhibitors). Chromatin was incubated with anti-HA magnetic beads (Pierce) at 4 °C overnight with gentle rotation. On a magnetic stand, the beads were washed twice with wash buffer 1 (50 mM Tris HCL pH 7.4, 1 M NaCl, 1 mM EDTA, 1 % NP-40, 0.1 % SDS, 0.5 % Na Deoxycholate plus protease inhibitors) and once with wash buffer 2 (20 mM Tris HCL pH 7.4, 10 mM MgCl2, 0.2 % Tween-20 plus protease inhibitors). The beads in wash buffer 2 were transferred to a new 1.5 mL Eppendorf tube and placed on a magnetic stand to remove the wash buffer. Decrosslinking was carried out in 1X TE plus 1 % SDS, proteinase K, and 400 mM NaCl at 65 °C for 4 h, followed by phenol, chloroform, and isopropyl alcohol precipitation of the DNA. Libraries were constructed using NEBNext Ultra II NEB Library Prep Kit and NEBNext Multiplex oligos (Illumina).
GEO
GSM9218576_r1