GSM4680362: Pat21_3: HBEC-ALI nonCF21 rep3; Homo sapiens; RNA-Seq
For RNA isolation 12 mm Transwell inserts (Corning) were placed on ice in a 6-well plate. 300 µl TRizol (Sigma) was added directly on the insert. The cells were then scraped and transferred to a 1.5 ml tube for 5 min at room temperature, 60 µl chloroform (Sigma) was added and the tube was shaken for 15 sec. After 10 min at room temperature the tubes were centrifuged at 12,000 x g for 20 min at 4°C. 180 µl isopropanol was added to the aqueous phase for 5-10 min at room temperature and centrifuged at 12,000 x g for 10 minutes at 4°C. The RNA pellet was washed with 300 µl of 70% cold EtOH, centrifuged at 11,400 rpm for 5 min at 4°C and dissolved in 25 µl DEPC (Sigma) water. The quality of the RNA was checked using a 2100 Bioanalyser (Agilent) and the samples were stored at -80°C before analysis Stranded sequencing libraries of poly-adenylated mRNA were prepared from 200 ng of isolated total RNA using the Stranded mRNA Prep (Illumina) according to the manufacturer's recommendations. In short, poly-adenylated mRNA was enriched using magnetic oligo-dT beads. The enriched mRNA was then fragmented enzymatically and the first and second strand cDNA syntheses were performed. In the synthesis of the second strand, dUTPs are incorporated instead of dTTP, which will not be amplified in future PCR reactions. To the resulting double stranded cDNA, adapters were ligated and the stranded library is amplified and sequenced on an HiSeq2500 sequencer (Illumina).
GEO Accession
GSM4680362