GSM4506081: Ctrl_RNA-seq_rep2; Mus musculus; RNA-Seq
For RNA isolation, total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's recommendations. reverse transcription was carried out using the TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). For ChIP, In accordance with the manufacturer's protocol, a SimpleChIP™ Enzymatic Chromatin IP Kit (CST) was used for the ChIP assays. Briefly, formaldehyde cross-linked chromatin was obtained from about 2×107 mESCs. Cross-linked chromatin was immunoprecipitated with antibodies to H3K4me1, H3K4me3, H3K27me3, H3K27ac, H3K9me3, gamma-phosphorylated H2AX and CTCF. Non-specific mouse IgG and H3 were used for negative and positive controls, respectively. For RNA-seq, briefly, total RNA was treated with the Epicentre Ribo-Zero kit to remove the all the rRNAs. The remaining RNAs were processed using the TruSeq RNA Sample Prep Kit according to the Illumina protocol. Then, RT-PCR was performed with Phusion High-Fidelity DNA polymerase, Index (X) Primer, and Universal PCR primers. Finally, the products were purified using the AMPure XP system, and library quality was evaluated on an Agilent Bioanalyzer 2100 system. The RNA library was sequenced on an Illumina Hiseq 4000 platform, and 150 bp paired-end reads were generated. For ChIP-seq, DNA libraries of H3K4me1,H3K27ac, H3K4me3, H3K27me3, H3K9me3, phospho S139 gamma H2A.X and CTCF were prepared for HiSeq 4000 sequencing as previously before.
GEO Accession
GSM4506081