title: GSM4668496: Hippocampus WT1; Mus musculus; RNA-Seq
library_construction_protocol: Hippocampus was removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated sing 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-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.
attributes.tag: GEO Accession
attributes.value: GSM4668496