GSM6607088
A total amount of 1 µg of RNA per sample was used as the input material for the RNA sample preparations. Sequencing libraries were generated using an NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out by using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5x) or by using sonication with a Diagenode Bioruptor Pico for fragmentation of RNA strands. First-strand cDNA was synthesized using random hexamer primers and M-MuL V 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 activity. After adenylation of the 3' ends of the DNA fragments, NEBNext adaptors with a hairpin loop structure were ligated to prepare the fragments for hybridization. To preferentially select cDNA fragments 150-200 bp in length, the library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 µI of 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. Finally, the PCR products were purified (AMPure XP system), and library quality was assessed on an Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using a PE Cluster Kit cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the libraries were sequenced on an Illumina platform, and paired-end reads were generated (Novogene).