title: GSM8741466: CD34_SP140i_Rep2; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was harvested in trizol (Invitrogen), and extracted by NovoGene. RNA integrity was assessed using the RNANano 6000 Assay Kit of the Bioanalyzer2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material for the RNA sample preparations. 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 First Strand SynthesisReaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-). Second strand cDNAsynthesis was subsequently performed usingDNA PolymeraseI and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3'ends of DNA fragments, 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 (Beckman Coulter, Beverly, USA). 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 Bioanalyzer2100 system.
identifiers.namespace: GEO
identifiers.id: GSM8741466_r1