GSM2056022: SSM_FLI_Shiva9_1; Homo sapiens; RNA-Seq
Total RNA was isolated using Qiazol (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, with modifications. In brief, the fibroblasts were pelleted in 2 ml safe-lock tubes (Eppendorf, Hamburg, Germany). 1 ml cooled Qiazol and one 5 mm stainless steel bead (Qiagen) were added. Homogenization was performed using a TissueLyzer II (Qiagen) at 20 Hz for 1 min. After incubation for 5 min at RT, 200 ml chloroform was added. The tube was shaken for 15 sec and incubated for 3 min at RT. Phase separation was achieved by centrifugation at 12,000 x g for 20 min at 4oC. The aqueous phase was transferred into a fresh cup and 10 mg of glycogen (Invitrogen, Darmstadt, Germany), 0.16 volume NaOAc (2 M, pH 4.0) and 1.1 volume isopropanol were added, mixed and incubated for 10 min at RT. The RNA was precipitated by centrifugation with 12,000 x g at 4oC for 20 min. The supernatant was removed and the pellet was washed with 80% ethanol twice and air dried for 10 min. The RNA was re-suspended in 20 l DEPC-treated water by pipetting up and down, followed by incubation at 65oC for 5 min. The RNA was quantified with a NanoDrop 1000 (PeqLab, Erlangen, Germany) and stored at -80oC until use. Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit v2 following the manufacturer’s instruction.
GEO Accession
GSM2056022