title: GSM5235837: A; Homo sapiens; RNA-Seq
library_construction_protocol: A total amount of 3 μg RNA per sample was used as input material for the RNA sample  preparations. Firstly, ribosomal RNA was removed by Epicentre Ribo-zero™ rRNA  Removal Kit (Epicentre, USA), and rRNA free residue was cleaned up by ethanol  precipitation. Subsequently, sequencing libraries were generated using the rRNA-  depleted RNA by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®  (NEB, USA) following manufacturer's recommendations. Briefly, fragmentation was  carried out using divalent cations under elevated temperature in NEBNext First Strand  Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random  hexamer primer and M-MuLV Reverse Transcriptase(RNaseH-). Second strand cDNA  synthesis was subsequently performed using DNA Polymerase I and RNase H. In the  reaction buffer, dNTPs with dTTP were replaced by dUTP. Remaining overhangs were  converted into blunt ends via exonuclease/polymerase activities. After adenylation of  3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated  to prepare for hybridization. In order to select cDNA fragments of preferentially  150~200 bp in length, the library fragments were purified with AMPure XP system  (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used  with size-selected, adaptor-ligated cDNA at 37° C for 15 min followed by 5 min at  95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, products were  purified (AMPure XP system) and library quality was assessed on the Agilent  Bioanalyzer 2100 system.