title: GSM8410430: leaves,  PagSUPa overexpression (OE) line 7 , rep4; Populus alba x Populus glandulosa; RNA-Seq
library_construction_protocol: 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 SMARTScribe™ 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. the mRNA was hybridized with Oligo（dT）probe and Captured by Magnetic Beads. The mRNA was fragmented by high temperature，then reverse-transcripted to first strand DNA. First strand DNA was used as template to synthesis second strand DNA, obtaining dsDNA. The dTTP tailed adaptor was ligated to both ends of the dsDNA fragments. The ligation product was amplified by PCR, and circularized to get single-stranded circular(ssCir) library. The ssCir library was then amplified through rolling circle amplification (RCA) to obtain DNA nanoball(DNB).