GSM2441396: nuclear mRNA in knockdown-FLII HBE; Homo sapiens; RNA-Seq
The RNC extraction was performed as described by Esposito et al. (18) with modifications. In brief, cells were pre-treated with 100 mg/ml cycloheximide for15 min, followed by pre-chilled phosphate buffered saline washes and addition of 2 ml cell lysis buffer [1%Triton X-100 in ribosome buffer (RB buffer) [20mM HEPES-KOH(pH=7.4), 15 mM MgCl2, 200 mM KCl, 100 mg/ml cycloheximide and 2 mM dithiothreitol]. After 30-min ice-bath, cell lysates were scraped and transferred to pre-chilled 1.5 ml tubes. Cell debris was removed by centrifuging at 13200 g for 10 min at 4°C. Supernatants were transferred on the surface of 20 ml of sucrose buffer (30%sucrose in RB buffer). RNCs were pelleted after ultra-centrifugation at 185 000 g for 5 h at 4°C. RNC-RNA was purified using Trizol method. Libraries were prepared according to NEBNext® mRNA Library Prep Master Mix Set for Illumina®.
GEO Accession
GSM2441396