RNA-seq of HEK293: control 1repl
RNA was extracted by adding 0.1 volume of 10% SDS, one volume of acidic phenol-chlorophorm preheated to 65¡C and incubated at 65¡C for 5 min. The reaction was cooled on ice for 5 min. Phases were separated by centrifugation at 21,000xg for 5 min. Equal volume of acid phenol-chloroform was added to the aqueous phase, separated by centrifugation and supplemented with an equal volume of chloroform Ð isoamyl alcohol (24:1). Upon separation, the aqueous phase was supplemented with 0.1 vol 3M NaOAc (pH 5.5) and an equal volume of isopropanol. Samples were precipitated for 3h at -20¡C. RNA was pelleted at 21,000xg at 4¡C, and the dried pellets resuspended in DEPC-H2O. Spike-ins were added upon rRNA depletion using Ribo-Zero Magnetic Gold Kit and used to set the detection threshold. The rRNA-depleted samples were fragmented in alkaline fragmentation buffer, dephosphorylated and fragments ranging from 20-35 nt were size-selected on 15% polyacrylamide gel containing 8M urea. The adapters were ligated directly to the 5Õ- and 3Õ-ends, converted into cDNA libraries and sequenced.