GSM2628219: T5_10uM_6h; Homo sapiens; OTHER
Total RNA was extracted using an RNeasy Miniprep Kit (Qiagen). Poly(A) mRNA was purified using Oligotex -dT30<Super> mRNA Purification Kit (Takara Bio., Japan, Shiga). Poly(A) mRNA was fragmented at 70°C for 5 min using RNA fragmentation buffer (Life Technologies) and first strand cDNA was synthesised using SuperScript Double-Stranded cDNA Synthesis Kit (Life Technologies) and P7-t25-vn oligo-dT primer. Subsequently, second strand cDNA was synthesised and then end-repaired, at 30°C for 20 min, using NEBNext End Repair Module (New England Biolabs, Ipswich, MA, USA). cDNA was purified using MinElute Reaction Cleanup Kit (Qiagen) and 100 pmol annealed P5-splinkerette ligated with cDNA for 2 h at 16°C. Ligated cDNA was purified and size-selected (~220 bp), using NucleoMag NGS Clean-up and Size Select Kit (Takara Bio). Samples were amplified with P5 and P7 primers using Phusion Hot Start Flex 2X Master Mix (New England Biolabs). After purification, the library was sequenced on the Illumina HiSeq 2000.
GEO
GSM2628219