title: GSM8833797: TM3_Py_rep3; Mus musculus; RNA-Seq
library_name: GSM8833797
library_construction_protocol: RNA was extracted using TRIzol reagent according to the manufacturer's instructions. RNA integrity was assessed with the RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 system (Agilent, 5067-1511).Total RNA was used as input material for RNA sample preparation. 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). 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. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, adaptors with hairpin loop structures were ligated for hybridization. To select cDNA fragments of 370–420 bp in length, the library fragments were purified using the AMPure XP system. PCR amplification was performed with Phusion High-Fidelity DNA Polymerase, Universal PCR primers, and Index (X) Primer. Finally, the PCR products were purified again using the 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 TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.
identifiers.namespace: GEO
identifiers.id: GSM8833797_r1