NextSeq 500 sequencing; BRAT1 Links Integrator and Defective RNA Processing with Neurodegeneration
BRAT1 Links Integrator and Defective RNA Processing with Neurodegeneration
Cells were harvested from respective cell cultures. Human fibroblasts were cultured in Minimum Essential Medium Eagle (MEM; Gibco) supplemented with 15 % fetal bovine serum (FBS; Gibco), 2 mM L-glutamine (Gibco), and the antibiotics penicillin (100 units/ml) and streptomycin (100 μg/ml) (Pen/Strep; Gibco) at 37°C. Human wild type osteosarcoma cells (U2OS) were cultured in Dulbecco's Modified Eagle's Medium-high glucose (DMEM-high glucose; Sigma) supplemented with 10 % FBS and the antibiotics Pen/Strep at 37°C. BRAT1 KO gene edited U2OS cell lines were prepared using Cas9 and guide sequences designed in CRISPR direct (https://crispr.dbcls.jp). Total RNA was isolated from 1×106 cells by miRNeasy Micro Kit (Qiagen) with an additional DNase I treatment, according to the manufacturer's instructions. Sequencing libraries were prepared with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian user manual (Takara Bio USA, Inc).