title: GSM8655210: HKDC1 LKO sample 17; Mus musculus; RNA-Seq
library_name: GSM8655210
library_construction_protocol: RNA from liver was extracted by trizol protocol A total amount of l ug RNAper sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNextRUltraTMRNA Library Prep Kit for lluminaR(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 tecmperature 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 l 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 ragments were purified with AMPure XP system (Beckman Coulter,Beverly,USA).Then 3 ul USER Enzyme (NEB, USA) was used with size-selected,adaptor-ligated cDNAat 37C for 15 min followed by 5 min at 95C before PCR.Then PCR was performed with Phusion HighFidelity DNA polymerase,Universal PCR primers and Index (X)Primer.Atlast,PCR products were purified (AMPure XPsystem) and library quality was assessed on the Agilent Bioanalyzer 2100 system.