title: GSM8017502: NC_C; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was harvested using Rneasy mini plus kit (Qiagen) following manufacturer's protocol. 1.3 μg of total RNA was used for the construction of sequencing libraries. Total RNA was used as input material for the RNA sample preparations. Sequencing  libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA,  Catalog #: E7530L) following manufacturer's recommendations and index codes were  added to attribute sequences to each sample.  Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads.  Fragmentation was carried out using divalent cations under elevated temperature in NEB  Next First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using  random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Second strand  cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H.  Remaining overhangs were converted into blunt ends via exonuclease/polymerase  activities. After adenylation of 3' ends of DNA fragments, NEB Next Adaptor with hairpin  loop structure were ligated to prepare for hybridization. In order to select cDNA fragments  of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP  system (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, PCR products were purified (AMPure XP system) and library  quality was assessed on the Agilent 5400 system（Agilent，USA） and quantified by QPCR  (1.5 nM).  The qualified libraries were pooled and sequenced on Illumina platforms with PE150  strategy in Novogene Bioinformatics Technology Co., Ltd (Beijing, China), according to  effective library concentration and data amount required.