title: GSM4260916: 02_Org_sh; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted with Trizol reagents (Thermo) following the manufacturer's instructions. 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 was synthesized using random hexamer primer and M-MuLVReverse 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 3 μg total RNA per sample was used as input material for the  small  RNA  library.  Sequencing  libraries  were  generated  using  NEBNext® Multiplex  Small  RNA  Library  Prep  Set  for  Illumina® (NEB,  USA.) following manufacturer's  recommendations  and  index  codes  were  added  to  attribute sequences  to  each  sample.  Briefly,  NEB  3'  SR  Adaptor  was  directly,  and  specifically ligated to 3' end of miRNA, siRNA and piRNA. After the 3' ligation  reaction,  the  SR  RT  Primer  hybridized  to  the  excess  of  3'  SR  Adaptor  (that remained free after the 3' ligation reaction) and transformed the single-stranded DNA  adaptor  into  a  double-stranded  DNA  molecule.  This  step  is  important  to prevent adaptor-dimer formation, besides, dsDNAs are not substrates for ligation mediated by T4 RNA Ligase 1 and therefore do not ligate to the 5´  SR Adaptor in the subsequent ligation step. 5´ends adapter was ligated to 5´ends of miRNAs,  siRNA  and  piRNA.  Then  first  strand  cDNA  was  synthesized  using  M-MuLV  Reverse  Transcriptase  (RNase  H–).  PCR  amplification  was  performed  using  LongAmp  Taq  2X  Master  Mix,  SR  Primer  for  illumina  and  index  (X)  primer. PCR products were purified on a 8% polyacrylamide gel (100V, 80 min). DNA  fragments corresponding to 140~160 bp (the length of small noncoding RNA plus the 3' and 5' adaptors) were recovered and dissolved in 8 μL elution buffer. At last, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips.
attributes.tag: GEO Accession
attributes.value: GSM4260916