title: GSM1467498: PNFF-hMPN1; Homo sapiens; RNA-Seq
library_construction_protocol: Hot phenol RNA dextraction protocol (Schmitt ME, Brown TA & Trumpower BL (1990) A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 18: 3091–3092); Trizol reagent was used to isolate total RNA from human cells. 10 µg of total RNA was ligated to Thaliana_lig_2 adaptor oligonucleotides (rArGratcggaagagcgtcgtgtagggaaagag-ddT where r denotes ribonucleotides) using Thaliana tRNA ligase. RNA was purified and treated with 20 U of rSAP (Affymetrix) in rSAP buffer (25 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 mM ATP (NEB)) at 37°C for 1 hour in order to remove 2' phosphates remained on ligated RNA. RNA was fragmented with RNA fragmentation reagent (Ambion). The fragmented RNA was then reverse transcribed using primers complementary to the ligated RACE adaptor. the obtained cDNA was used to construct sequencing library.