GSM8746090: GE2; Mus musculus; RNA-Seq
SMARTer cDNA synthesis starts with picogram amounts of total RNA or single cell/several cells. A modified oligo(dT) primer (the SMART CDS Primer) primes the first-strand synthesis reaction. When SMARTScribeTM Reverse Transcriptase reaches the 5' end of the mRNA, the enzyme's terminal transferase activity adds a few additional nucleotides to the 3' end of the cDNA. The carefully-designed SMARTer Oligonucleotide base-pairs with the non-template nucleotide stretch, creating an extended template to enable SMARTScribe RT continue replicating to the end of the oligonucleotide. The resulting full-length, single-stranded (ss) cDNA contains the complete 5' end of the mRNA, as well as sequences that are complementary to the SMARTer Oligonucleotide. Amplify sscDNA by LD PCR and get enough dscDNA for library construction. cDNA was fragmented by dsDNA Fragmentase (NEB, M0348S) by incubate at 37°C for 30min. Library construction begins with fragmented cDNA. Blunt-end DNA fragments are generated using a combination of fill-in reactions and exonuclease activity, and size selection is performed with provided sample purification beads. An A-base is then added to the blunt ends of each strand, indexed Y adapters are ligated to the fragments, and the ligated products are amplified with PCR. And then we performed the paired-end sequencing on an Illumina NovaseqTM 6000 at the (LC Sceiences, USA) following the vendor's recommended protocol.