GSM6383292: CON-NEG-90; Mus musculus; RNA-Seq
Blood RNA from 6–7 individual mice per group was used in this study. Male mice submandibular blood was collected using 20-G hypodermic needles into EDTA-coated tubes. The tubes were centrifuged at 1000xg for 20 min at 4°C, and 100 μl of the blood cell fractions were put in RNAprotect animal blood tubes (Qiagen,) and stored at -80°C. Total RNA, including miRNAs, was extracted using RNeasy Protect animal blood kit (Qiagen). RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies) and RNA integrity was checked using 4200 TapeStation. rRNA depletion along with globin depletion was performed using QIAGEN FastSelect rRNA HMR/Globin Kit. RNA-seq library preparation used NEBNext Ultra II RNA Library Prep Kit for Illumina by following the manufacturer's recommendations. Briefly, enriched RNAs were fragmented for 15 min at 94°C. The first and second cDNA strands were subsequently synthesized. cDNA fragments were end-repaired and adenylated at 3'-ends, and a universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using Agilent Tapestation 4200 and quantified using Qubit 2.0 Fluorometer. Sequencing libraries were multiplexed and clustered on the Illumina HiSeq instrument according to the manufacturer's instructions. The samples were sequenced using a 150 paired end configuration.