GSM8426411: RNAseq_MYCNER_shTCEAL1_OHT_Dox_rep3; Homo sapiens; RNA-Seq
For ChIP and ChIP-Rx sequencing, cells were fixed with formaldehyde and spiked with murine NIH-3T3 cells at a 1:10 cell ratio. The crosslinked chromatin was extracted and fragmented using a Covaris Ultrasonicator. Chromatin was then added to a pre-incubated bead/antibody mix. Following incubation and stringent washing, chromatin was eluted, protein and RNA were digested with proteinase K and RNase A and DNA was isolated by phenol-chloroform. For RNA sequencing, cells were harvested and total RNA was extracted with RNeasy MiniKit (Qiagen) according to manufacturer's instructions. On-column Dnase I digestion was performed followed by mRNA isolation with the NEBNext Poly(A) mRNA Magnetic Isolation Kit (NEB). ChIP and ChIP-Rx libraries were prepared with the NEBNext Ultra II DNA Library Prep kit. RNAseq libraries were generated with the NEBNext Ultra II Directional RNA Library Prep kit. All libraries were PCR-amplified using a set of Illumina compatible index primers. For all described libraries, a qPCR was performed prior to the indexing-PCR, to determine optimal PCR cycle numbers. Following the indexing-PCR, libraries were purified using SPRI-beads (Backman Coulter). Library quality and fragment size distribution was analyzed on a Fragment Analyzer (Advanced Analytical). Sequencing was performed in single-read or paired-end mode on a NextSeq2000 platform (Illumina).