BAF250B-associated SWI/SNF chromatin-remodeling complex is required to maintain undifferentiated mouse ES cells

We used BAF250b-/- ES cells, two independently derived clones, at 18 and 72 hr in culture and compared them with parental cell line (wild-type) at the same time in culture (two replications each). Total RNAs were extracted using Trizol (Invitrogen) according to manufacturer protocol. 2.5 ug of total RNA samples were labeled with Cy3-CTP using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent). A reference target (Cy5-CTP-labeled) was prepared from the Universal Mouse Reference RNA (Stratagene). Labeled targets were purified using an RNeasy Mini Kit (Qiagen) according to the Agilent's protocol, quantitated by a NanoDrop scanning spectrophotometer (NanoDrop Technologies), and hybridized to the NIA Mouse 44K Microarray v2.2 (whole genome 60-mer oligo; manufactured by Agilent Technologies, #014117) (Carter et al. 2005) according to the Agilent protocol (G4140-90030; Agilent 60-mer oligo microarray processing protocol - SSC Wash, v1.0). All hybridizations were carried out in the two color protocol by combining one Cy3-CTP-labeled experimental target and Cy5-CTP-labeled reference target. Microarrays were scanned on an Agilent DNA Microarray Scanner, using standard settings, including automatic PMT adjustment. Keywords: genetic modification design,time series design Biological replications were performed on BAF250b-/- ES cells with their parental line (wild-type) at 18 and 72 hrs after passaging, as well as with 12 other pluripotent cells (ES and EG cells from strains C57BL/6 and 129 data from Sharova et al., 2007) using whole-genome mouse microarrays (NIA 44k V2.2 with 60-mer synthesized oligos).