GSM5776077: KO_pDC_1; Mus musculus; RNA-Seq
Total RNA of 3 -10 x 10^5 cells was extracted with the TRIzol reagent. BGI-Tech (Shenzhen, China) constructed the mRNA library and performed sequencing. Briefly, total RNA was treated with DNase I and RNase H to remove DNA and rRNA. Purified mRNA was fragmented and subjected to first-strand and second-strand synthesis. A-Tailing Mix and RNA Index Adaptors were added to beads-purified reaction product for end repair. PCR was used to amplify cDNA fragments with adaptors, and Ampure XP Beads were used to purify product. Library was QC-checked with Agilent 2100, heat-denatured, and circularized by the splint oligo sequence to generate single strand circle DNA. This final library was rolling circle-amplified with phi29 DNA polymerase (Thermo) to produce DNA nanoballs, and more than 300 copies were generated for one molecule. DNA nanoballs were loaded into the patterned nanoarray, and the BGISEQ-500 platform (BGI-Shenzhen, China) was used to generate single end 50 bases reads.