title: GSM6635128: AKO-BAT rep1; Mus musculus; RNA-Seq
library_name: GSM6635128
library_construction_protocol: Total RNA from each sample was extracted using RNAiso Plus (Takara Biotech), and concentration was measured using an ND-1000 NanoDrop (Thermo Scientific, Wilmington, DE). A total amount of 1 μg RNA per sample 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 Synthesis Reaction Buffer(5X). Firststrand cDNA wassynthesized 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, 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 Bioanalyzer 2100 system.