GSM5531930: Cbx8_LOVO_UNC7040_rep1; Homo sapiens; ChIP-Seq
For RNA-seq sample preparation, 5-10x10^6 SUDHL-4 or LoVo cells (untreated, UNC7263 or UNC7040 treated at least in duplicate) were trypsinized and collected by centrifugation for 5 min at 500 g. Subsequently, cell pellets were washed 1× with PBS and collected in 1X DNA/RNA protection reagent (Monarch Total RNA Miniprep Kit, NEB). Cells were lysed and total RNA was isolated following the mammalian cell protocol inlcuding on-column DNase I treatment. Total RNA samples were submitted to Novogene Co. for quality control and library preparation, applying poly-A enrichment and using the NEBNext Ultra II RNA Library Prep kit (NEB), indexed using NEBNext Multiplex Oligos (NEB). Final libraries were sequenced as 150 bp paired-end reads on the Illumina HiSeq platform. ChIP-seq Library PreparationLibraries were prepared using the NEXTflex ChIP-Seq kit (Bio Scientific) following the “No size-selection cleanup” protocol. Each sample of ChIPed DNA was end-repaired and ligated to unique barcoded adaptors to produce individual libraries. Libraries corresponding to samples to be directly compared to each other (e.g. DMSO vs UNC7040) were pooled together and purified using 1 volume of Agencourt AMPure XP (Beckman Coulter). The pooled libraries were eluted with 23 µL of elution buffer (NEXTflex ChIP-Seq kit) and amplified using the KAPA Real-Time Library Amplification Kit (KAPABiosystems) following the kit instructions. Finally, the amplified libraries were size-selected and purified using 0.9x volume of Agencourt AMPure XP (Beckman Coulter). Library quality control including determination of average size and concentration was performed prior to sequencing by commercial Next Generation Sequencing providers. NGS libaries were eventually sequenced as 150 bp paired-end reads on the Illumina HiSeq platform. NGS libaries were eventually sequenced as 150 bp paired-end reads on the Illumina HiSeq platform.