title: GSM3855589: w-B-W-4-JR; Pelophylax nigromaculatus; RNA-Seq
library_construction_protocol: Total RNA of each sample was extracted and purified using Trizol (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. After purified with poly-T oligo-attached magnetic beads, the mRNAs were fragmented. First-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H−). Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3′ ends of the DNA fragments, NEBNext Adaptors with a hairpin loop structure were ligated to prepare for hybridization. To preferentially select cDNA fragments of 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 a Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) primer. Finally, PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq 2500 platform by NovoGene (Beijing), and paired-end reads were generated.
attributes.tag: GEO Accession
attributes.value: GSM3855589