Rna-seq of mouse musculus : audlt male brain
RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturers instructions (Illumina, San Diego, CA). The RNA-seq transcriptome librariy was prepared following SMART-Seq_V4 Ultra Low Input RNA Kit for Sequencing from Clontech (San Diego, CA) using 10ng of total RNA. Reverse transcription (onestrand synthesis) was performed first, and RNA with a polyA tail (major mRNA) was reverse transcribed using Oligo(dT) primer. Three cytosine (C) residues are added to the 3' end of the cDNA strand because of the use of a special active reverse transcriptase (MMLVRT) for reverse transcription. Two strands of cDNA were synthesized with the use of TSO (template-switching oligo) primers, thereby replacing RNA complementary to one strand of cDNA.Then, the cDNA was expanded to ng level by PCR amplification. DNA disruption using a modified, highly active Tn5 transposase was performed while the linker was added to both ends of the cDNA. After the last PCR amplification, the machine was ready for sequencing. After quantified by Qubit 4.0, paired-end RNA-seq sequencing library was sequenced with the NovaSeq X plus sequencer (2  150bp read length).