GSM6703423: breast, 1-1; Mus musculus; RNA-Seq
Total RNA was isolated using TRIzol (Invitrogen) and miRNeasy mini kit (QIAGEN) according to manufacturer's instructions, which efficiently recovered all RNA species, including miRNAs. RNA quality and quantity was measured by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies) and RNA Integrity was determined by gel electrophoresis 1) RNA product after fragmentation mRNA enrichment (or rRNA removal) treatment. 2) Reverse transcription to generate first-strand cDNA: 3) Synthesis of double-stranded cDNA by dUTP addition: 4) End repair of double-stranded cDNA by each polymerase: 5) Ligase ligation of Illumina-specific connectors. (6) PCR amplification and purification to obtain the final RNA sequencing library. The constructed libraries were quality checked by Agilent 2100 Bioanalyzer (library concentration, fragment size 400-600bp, presence of splice, etc.) and final quantification of the libraries was performed by 9PCR method. The libraries were mixed according to the quantification results and the final sequencing data volume required to enter the sequencing process.