GSM5563240: G401 MAK683 rep2; Homo sapiens; RNA-Seq
RNA samples were harvested with Trizol reagent. Total RNA 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.Ribosomal RNA (rRNA) is depleted for target transcript enrichment.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(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 250~300 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.A total amount of 50 ng DNA per sample was used as input material for the Chip sample preparations. Sequencing libraries were generated using NEBNext Ultratm DNA Library PrepKit for Illumina(NEB USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly,the Chip DNA was purified using AMPure XP system (Beckman Coulter Beverly,USA). After adenylation of 3'ends of DNA fragments, the NEB Next Adaptor with hairpin loop structure were ligated to prepare for hybridization. Then electrophoresis was used to select DNA fragments specified in length. 3 uL USER Enzyme(NEB，USA) was used with size-selected,adaptor-ligated DNA at 37C for15 min followed by5 min at 95C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index(X) Primer. At last. PCRproducts were purified(AMPure XP system) and library quality was assessed on the AgilentBioanalyzer 2100 system.