GSM2571068: polyA.enriched.NONT.rep1; Homo sapiens; RNA-Seq
For ChIP-seq, cells were crosslinked with 1% paraformaldehyde for 10 minutes at room temperature with gentle rotation, and then quenched by 0.125 M glycine solution. After washing, nuclei were sonicated using Covaris Sonicator, and the supernatant was used for immunoprecipitation with the indicated antibody. ChIP-sequencing libraries were prepared with Illumina's Tru-seq DNA sample prep kit. Total RNA was extracted from cells using RNeasy mini Kit (Qiagen) followed by ribosomal RNA depletion with the RiboZero kit (Epicenter). Poly(A) depleted and Poly(A)-enriched RNA were separated by 3 rounds of Oligo(dT) magnetic beads (Thermo). For sequencing, 2 μg of resulting RNA was used for ribosomal RNA depletion with the RiboZero kit (Epicenter) and libraries were made with the TruSeq RNA sample Prep Kit (Illumina). ChIP-sequencing libraries were prepared with Illumina's Tru-seq DNA sample prep kit using standard protocols. RNA-seq libraries were prepared with Illumina's TruSeq RNA sample Prep Kit using standard protocols. 4C-seq was performed following the protocol published in (van de Werken et al. 2012). In brief, cross-linked chromatin was digested with DpnII, ligated under diluted conditions. Cross-links were reversed and DNA was further digested by NlaIII and again ligated under diluted conditions. For each viewpoint approximately 3.2 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina adaptor sequence. Multiplexed samples were sequenced on the Illumina Nextseq500 system using 75 bp single-end reads according to the manufacturer's specifications.