The spleen was collected and smashed on ice. Total RNA samples were harvested using Trizol reagent. We use Agilent 2100 Bio analyzer (Agilent RNA 6000 Nano Kit) to do the total RNA sample QC: RNA concentration, RIN value,28S/18S and the fragment length distribution. 1. mRNA enrichment: Oligo dT Selection or rRNA depletion. 2. RNA fragment and reverse transcription: Fragment the RNA and reverse transcription to double-strand cDNA (dscDNA) by N6 random primer. 3. End repair, add A tailing and adaptor ligation:The 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 amplification:The ligation products were purified and many rounds of PCR amplification were performed to enrich the purified cDNA template using PCR primer. 5. Denature and cyclization:Denature the PCR product by heat and the single strand DNA is cyclized by splint oligo and DNA ligase. 6.Sequencing on BGISEQ- 500 platform.
GEO Accession
GSM3265181