title: GSM5005630: control input rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: For immunoprecipitation, on day 1 antibodies were diluted in RIPA-0.3 and bound to Dynabeads protein A (Thermo, cat. no. 10002D) by incubating at 4 °C for 3 hours. Afterwards, the bead-antibody complexes were washed twice with RIPA-0.3 and then incubated with sonicated chromatin at 4 °C overnight. On day 2, after 2 washes with RIPA-0.5, 1 wash with RIPA-0.3, 1 wash with RIPA-0, 2 washes with LiCl buffer (10 mM Tris-HCl, 1 mM EDTA, 0.25 M LiCl, 0.25% NP-40, and 0.25% NaDOC, pH 7.4), and 2 washes with TE buffer, bound protein-DNA complexes were resuspended in elution buffer (10 mM Tris-HCl, 1mM EDTA, 0.2 M NaCl, and 1% SDS, pH 7.4) supplemented with 10 µg/ml RNase A for both elution and RNA digestion, and incubated at 55 °C for 1 hour. Then Proteinase K was added to a final concentration of 200 µg/ml, and after 30 min incubation the temperature was increased to 65 °C for crosslink reversal. After incubation for 4 to 6 hours, DNA was purified by ChIP DNA Clean & Concentrator (Zymo Research, cat. no. D5205).