GSM6605984
Total RNA was extracted using QIAzol lysis reagent (QIAGEN). Isolate polyA RNA using dynabeads mRNA purification kit (Life Technologies). Fragmentation/The first cDNA systhesis (SuperScript II reverse transcription kit)/The second-strand synthesis (Rnase H (Life Techonologies)/DNA polymerase (Enzymatics)/ Short double-stranded cDNA fragments purification: QIA quick PCR purification kit and Agencourt AMPure beads/end-repair with klenow polymerase (T4 DNA polymerase and T4 polynucleotide kinase)/ A single 3' A adding to the double-stranded cDNA (Kelnow (Enzymatics) and dATP (GE Healthcare)/The illumina PE Adapter oligo mix ligation/library amplification/denaturation of library with sodium hydroxide and diluted to 2.5 pM in hybridization buffer for loading into a HiSeq flowell.