title: Bulk RNA-seq of murine microglia: Sepp1_Sh2-6
design: Stranded mRNA Library construction procedure are listed below:1) mRNA enrichment and purification:Oligo dT Selection (mRNA enrichment): Use oligo dT beads to enrich mRNA with poly A tail.2) RNA fragment and reverse transcriptionFragment the RNA and first-strand cDNA was generated using random N6-primed reverse transcription, followed by a second-strand cDNA synthesis with dUTP instead of dTTP.3) End repair, add A and adaptor ligationThe synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated cDNA fragments;4) PCR amplificationPrior to PCR amplification, the dUTP-marked strand is selectively degraded by Uracil-DNA-Glycosylase(UDG). The remaining strand is amplified to generate a cDNA library suitable for sequencing. Many rounds of PCR amplification were performed to enrich the purified cDNA template using PCR primer;5) Single strand separation and cyclizationDenature the PCR product by heat and the single strand DNA is cyclized by splint oligo and DNA ligase;6) DNA nanoball synthesis7) Sequencing on DNBSEQ (DNBSEQ Technology) platform