GSM5708542: M2 Un 3; Homo sapiens; RNA-Seq
Cells were immediately treated with 300µL RNAProtect (Qiagen), collected into sterile RNAse-free tubes, and pelleted by centrifugation at 400 x g for 5 minutes using a MicroCL 21R microcentrifuge. Cells were lysed using 350µL of lysis buffer from an RNAeasy Plus Mini kit (Qiagen), with added β-mercaptoethanol, and lysate was stored at -80°C RNA was isolated from MDM lysates stored at -80°C using an RNAeasy Plus Mini kit (Qiagen) with DNAse treatment (RNAfree DNAse kit, Qiagen). RNA quality met sequencing standards upon analysis on an RNA 6000 Nano Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies). From total RNA, mRNA was isolated using polyA enrichment with d(T) beads (New England Biolabs), and strand-specific cDNA libraries were created using a KAPA RNA HyperPrep kit (Roche cat. no.: 07277253001). Library quality was assessed with an Agilent 2100 Bioanalyzer using a High Sensitivity DNA chip (Agilent). Libraries were sequences on an Illumina HiSeq2500.