title: GSM8729379: GC muscle_KO_04; Mus musculus; RNA-Seq
library_name: GSM8729379
library_construction_protocol: Flash-frozen muscles were thawed on ice and homogenized in QIAzol Lysis Reagent (Qiagen, 79306) using a bead homogenizer. RNA was isolated using the RNeasy Mini kit (Qiagen, 74106) per manufacturers recommendations with on-column DNAse digestion. Integrity of RNA samples was evaluated using the Agilent 2100 bioanalyzer. 1 μg of total RNA per sample was used as input material for RNA sample preparations. Sequencing libraries were generated using 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 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, adaptorligated 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.
identifiers.namespace: GEO
identifiers.id: GSM8729379_r1