Small RNA-Seq of mouse mammary gland: sample 3 of day 12 of lactation
Total RNA was extracted from mammary gland tissue samples using TRIzol reagent (Invitrogen) according to the manufacturers instructions. Subsequently, RNA purity was determined using a Nano Drop ND2000 spectrophotometer (Thermo Fisher Scientific) at 260 and 280 nm, and the RNA integrity number (RIN) was assessed with an Agilent 2100 Bioanalyzer (Agilent). To construct the library, about 3 g of total RNA was extracted from each sample. Total RNA was separated on a 15% urea denaturing polyacrylamide gel electrophoresis (PAGE) gel, and small RNA regions corresponding to the 18-30 nt bands in the labeled lane (14-30 ssRNA Ladder Marker, TAKARA) were excised and recovered. Subsequently, the isolated small RNAs were linked with adenylated 3 adapters annealed to unique molecular identifiers (UMI), followed by 5adapter ligation. The adapter-ligated small RNAs were then reverse transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen), and the cDNA fragments were enriched using reverse transcription-polymerase chain reaction. The cDNA fragments were selected for target fragments of 110~130 bp using agarose gel electrophoresis, and then purified using QIAquick Gel Extraction Kit (QIAGEN). The DNBseq platform (BGI) was used to sequence the final cDNA.