title: GSM4715093: non-treated control RNA-seq for high-low comparison, rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA of cells was isolated using RNeasy Mini Kit (Qiagen), and cDNA were synthesized using PrimeScript first strand cDNA synthesis kit (Takara) with oligo-dT. The RNA-seq libraries were constructed by BGI, Shenzhen, as the platform BGISEQ-500 instruction described. ChIP-seq were performed as previously described (Xiong et al., Molecular Cell 64, 913-925). The ChIP-seq libraries were constructed using NEBNext DNA Sample Prep Master Mix (NEB). DNA libraries were subjected to size-selection using AMPure XP beads (Beckman Coulter) and PCR amplification (18-20 cycles for ChIP-seq DNA libraries). The NEBNext Enzymatic Methyl-seq Conversion Module (NEB E7125L) was used for measuring genome-wide DNA methylation level. Purified genomic DNA containing 0.05% CpG methylated pUC19 control DNA and 1% unmethylated lambda DNA was sheared to a mean length of 450 bp with Covaris M220 Focused-ultrasonicator. End repair, A-tailing, methylated adaptor ligation and size selection were performed on 125 ng fragmented genomic DNA with Kapa hyper prep kit (Kapa Biosystems KK8504) according to the manufacturer's instructions. The methylated adaptor ligated DNA fragments were then treated with oxidation of 5mC and 5hmC, clean-up of TET2 converted DNA, denaturation of DNA, deamination of cytosines and clean-up of deaminated DNA using the NEBNext Enzymatic Methyl-seq Conversion Module (NEB E7125L). The deaminated DNA was amplified with KAPA HiFi Hotstart Uracil+ ReadyMix PCR kit using primers from NEBNext multiplex oligos for Illumina (index primers set 1) (NEB E7335) (Kapa Biosystems KK2801) according to the manufacturer's protocol. PCR cycling condition was as follows: 98℃ for 45 s, followed by 7 cycles of 98℃ for 15 s, 60℃ for 30 s, 72℃ for 30 s, and final extension 72℃ for 1 min, then hold at 4℃. Libraries were sequenced on an Illumina NovaSeq 6000 using 150-bp paired-end mode.
attributes.tag: GEO Accession
attributes.value: GSM4715093