Total RNAs were extracted from the corpus callosum dissected from 8- to 10-week old wild-type and Bmal1-/- mice as well as primary neurospheres cultured from  8- to 10-week old wild-type and Bmal1-/- mice using TRIzol. 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
GSM3966396