GSM3402620: D2 SMARTA TH1, biological rep3 [ATAC-seq]; Mus musculus; ATAC-seq
ATAC-seq: 50,000 sorted cells were treated with lysis buffer and then centrifuged. The nuclei preparation was harvested after discarding the supernatant. Libraries were prepared with the Nextera DNA Sample Preparation Kit (FC-121-1030, Illumina). Briefly, the nuclei preparation was labeled with Nextera enzyme and immediately amplified by PCR of 9-10 cycles with barcoded primers and sequenced on NextSeq500 in a 150 bp/150 bp or 76 bp/76 bp paired end run. ChIP-seq: DNA-protein complexes of target cells were extracted and fixed with the Simple ChIP Enzymatic Chromatin IP Kit (Magnetic Beads) (#9003; Cell Signaling Technology) according to the manufacturer's instruction. Chromatin fragments were immunoprecipitated with anti-H3K27me3 (Cell Signaling Technology, #9733, Clone number: C36B11) with ChIP Grade Protein G magnetic Beads (#9006; Cell Signaling Technology). The Illumina's TruSeq indexed pair-ended DNA library preparation protocol was performed automatically on the SPRIworks system (Beckman Coulter). By using cartridge and method card specific to Illumina sequencing system, a fragment library can be prepared for Illumina sequencers. After individual libraries were constructed, qualities and band-sizes were assessed using Bioanalyzer High Sensitivity Chip (Agilent Technologies) and Qubit (Life Technologies). Libraries were also quantified by qPCR using the Library Quantification Kit for Illumina sequencing platforms (KAPA Biosystems), using an ABI 7900HT Real-Time PCR System (Life Technologies). Libraries were normalized to a working concentration of 10 nM, using the molarity calculated from qPCR and adjusted for fragment size with the Bioanalyzer analysis.
GEO Accession
GSM3402620