title: RNA-Seq of mus musculus
design: Briefly, to extract the total RNA, a total of 200 L serum sample were added with 1 mL QIAzol lysis reagent, vortexed well, and incu bated at room temperature for 5 min. Then, a total of 200 L chloroform/isoamyl alcohol  (24:1; v/v) were added to the sample, vortexed vigorously for 15 s, and incubated at room  temperature for 23 min. The samples were centrifuged at 12,000  g and 4 C for 8 min to collect the supernatant (5427R, Eppendorf Co., Ltd., Beijing, China), added with absolute  ethanol twice the volume of the supernatant, and the mixture was column purified and  washed once with 700 L RWT buffer and washed twice with 500 L RPE buffer (QIAgen, Beijing, China). The samples were centrifuged at 12,000  g and room temperature for 2  min with the purification column removed and replaced with a new collection tube, added with 20 L RNA free water, and incubated at room temperature for 1 min. Finally, the mixture was centrifuged at 12,000  g and room temperature for 2 min to elute RNA. The concentrations of eluted RNAs were determined with an Agilent 2100 Bioanalyzer (Agilent Technologies Co., Ltd., Beijing, China). The libraries for high-throughput miRNA  sequencing were constructed based on the segments of 1830 nt separated by PAGE gels (430 ssRNA Ladder Marker, TAKARA). The connections of both 3 and 5 adaptor systems and the RT-PCR analysis were performed by following the procedures provided by the manufacturers. The double stranded PCR products were denatured and then circularized by the splint oligo sequence to generate the single strand circle DNAs (ssCir DNAs), which were formatted as the final library. The final library was validated by the Agilent 2100 Bioanalyzer and amplified with phi29 to generate the DNA nanoball (DNB) with more than 300 copies of one molecule. The DNBs were loaded into the patterned nanoar ray to generate the single end 50 bp reads based on the combinatorial Probe-Anchor Synthesis (cPAS). The raw data were processed to generate the clean tags based on the manufacturers instructions. The clean tags were mapped onto the genome of Mus musculus with the reference version of GCF_000001635.26_GRCm38.p6 of the BioProject 320 PRJNA20689 at the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/assembly/; accessed on 21 April 2019) and further annotated based on miRbase database (http://www.mirbase.org/; accessed on 21 April 2019) and Rfam database (https://rfam.xfam.org/; accessed on 21 April 2019) with Bowtie2 .