GSM5919941: Panc1GR-NC2; Homo sapiens; RNA-Seq
GSM5919941
A total amount of 3 µg RNA per sample was used as input material for the RNA  sample preparations. Sequencing libraries were generated using NEBNext® UltraTM  RNA Library Prep Kit for Illumina® (NEB, USA) 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 NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA wassynthesized 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,  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, PCR products were purified (AMPure XP system) and  library quality was assessed on the Agilent Bioanalyzer 2100 system RNA library for lncRNA-seq was prepared as rRNA depletion and stranded method. After library construction, the concentration of library was measured by the Qubit® fluorometer and adjusted to 1ng/uL. Agilent 2100 Bioanalyzer was deployed to examine the insert size of the acquired  library. At last, the accurate concentration of cDNA library was again examined using qPCR. Once the  insert size and concentration of the library was identical, the samples can then be subjected for sequencing