GSM8743926: Naive (N050218-6); Mus musculus; RNA-Seq
RNA isolation was performed using the RNeasy Lipid Tissue Mini Kit (QIAGEN) with RNase-Free DNase Set (QIAGEN) to remove genomic DNA. The quality of RNA samples was determined by the Bioanalyzer 2100 system (Agilent Technologies). The RNA integrity number (RIN) of all our RNA samples used for RNA-seq was greater than 6.8. 2.1Library construction  Briefly, mRNA from Eukaryote organisms is purified from total RNA using poly-T oligo-attached magnetic beads (For prokaryotes, mRNA was purified through the removal of rRNA by using kit). The mRNA is first fragmented randomly by addition of fragmentation buffer. According to demand of customer, we have NEB library and strand specific library to choose.  NEB library: Then first strand cDNA is synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis is subsequently performed using DNA Polymerase I and RNase H. Double-stranded cDNA is purified using AMPure XP beads. Remaining overhangs of the purified double-stranded cDNA are converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure is ligated to prepare for hybridization(1). In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments are purified with AMPure XP system (Beckman Coulter, Beverly, USA). Finally, the final library is gotten by PCR amplification and purification of PCR products by AMPure XP beads.  Strand specific library: The construct method of strand specific library is similar to that of NEB library expect that when synthesizing the second strand cDNA, dTTP is replayed by dUTP. After overhangs of the purified double-stranded cDNA are converted into blunt ends, adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure is ligated to prepare for hybridization, the second strand cDNA is digested by USER enzyme. The following step is identical with NEB library construction. Below is the workflow chart: