GSM2665571: Day15_NKX2-1_3 NKX2-1_ChIP Seq; Homo sapiens; ChIP-Seq
For RNA sequencing, total RNA was collected from H9 hESCs and H9-derived neural derivatives, differentiated, at day 0, 4, 15, 25 and 35, using the Nucleospin RNA II (Takara) kit. This involves an on column DNA digestion to remove DNA contamination. RNA was quantified by NanoDrop ND-1000 (Thermo Scientific) and the quality of total RNA was further confirmed using the Agilent Bioanalyzer 2100. For ChIP sequencing, H9 hESC-derived MGE-like progenitor cells at day15 were dissociated with Accutase and the cell suspension was fixed with 1% formaldehyde and washed with ice cold PBS with protease inhibitors (Complete mini, Roche) . Pellet was lysed first with cell lysis buffer (5mM PIPES pH8, 85mM KCl, 0.5% NP-40) and then with nuclear lysis buffer (50mM Tris-HCl pH8.1, 10mM EDTA, 1% SDS). The lysed cell pellet was sonicated using the Sonifier 250 (Branson), the supernatant was collected after centrifugation at 14,000g, and the size of the sheared chromatin was confirmed to be between 300-500bp by agarose gel electrophoresis. For RNA-seq, library preparations and Illumina sequencing were performed by the Genome Technology Access Center (GTAC) at Washington University. For ChIP sequencing library preparation, 1ng of immunoprecipitated chromatin was used for Illumina sequencing-compatible library preparation with the DNA Smart ChIP-Seq kit (Takara) as recommended in the manufacturer’s protocol.
GEO
GSM2665571
GEO Accession
GSM2665571