GSM4699187: IOSE11; Homo sapiens; OTHER
Cell nuclei were extracted from fixed cell pellets and digested overnight with 375U of DpnII . After digestion, nuclei were resuspended in NEB buffer 2 (New England Biolabs) and restriction fragment overhangs were filled in with biotin-dATP using the DNA polymerase I, large Klenow fragment (incubated at 30oC for 2h). Proximity ligation was performed for 4 h at 16oC, then nuclei lysed and chromatin sheared for 9 min using the Covaris S220 Sonicator. For each sample, sheared chromatin was split into two tubes and incubated overnight with 4.6 mg of H3K27Ac antibody (Abcam, EP16602). The next day Protein A beads were used to capture H3K27Ac-associated chromatin which was eluted and purified with Zymo Research concentrator columns (columns were washed twice with 10 ml of water). The DNA concentration of the purified chromatin was to estimate the amount of TDE1 enzyme (Illumina) needed for tagmentation which was performed with biotin-labelled chromatin captured on streptavidin beads. Sequencing libraries were then generated by PCR of tagmented samples using the Nextera DNA preparation kit (Illumina) as per the manufacturer's instructions. Afterwards, size selection was performed using Ampure XP beads to capture 300-700 bp fragments. For each cell line, at least two independent sequencing libraries were pooled together to provide 25 ml of library at ≥10 nM for Illumina HiSeq4000
GEO Accession
GSM4699187