Illumina HiSeq 2500 sequencing; RNA-sequencing of human adherent glioblastoma stem cell line following RNA interference targeting TRIM26
RNA-sequencing of human adherent glioblastoma stem cell line following RNA interference targeting TRIM26
B67 GSCs were infected with MISSION TRC lentiviruses with shRNA targeting human TRIM26 (sh4050, sh4051) or with control (sh002) and 5 days later harvested for total RNA Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). Ribosomal RNA was removed by a hybridization method using Ribo-ZERO kits (Illumina).  mRNA was fragmented and reverse transcribed to yield double-stranded cDNA. cDNA was blunt ended, had an A base added to the 3â€² ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 cycles using primers incorporating unique index tags.