Mus musculus
Protocols: Serum Preparation Blood were collected by left ventricular or orbital puncture from male SD rats (200-250g, 8 weeks) and C57BL/6 mice (20-25g, 8 weeks). In order to prevent haemolysis, aspiration was performed slowly and evenly. Whole blood collected were left undisturbed for 1 h at room temperature (RT) and 2 h at 4℃, and then centrifuged at 1,000 g at 4℃ for 10 min. The resulting serum was transferred to a clean tube and centrifuged again at 2,000 g before aliquoted and stored at -80℃. Serums were thawed on ice. Every 4 to 5 samples (equal-volume) from the same species were pooled together. In the presnt study, the start volume of pooled serum for all sEVs isolation experiments was 500 µl. Each 500 µl pooled serum was centrifuged at 1,000 g at 4 °C for 15 min to remove debris, then 21,000 g at 4 °C for 15 min to remove larger EVs. The supernatant was filtered with a 0.22 μm filter (Millipore, Billerica, MA, USA). sEVs-enriched fractions (exosomes) were precipitated by ExoQuick (System Biosciences Inc., Mountain View, CA) according to the manufacturer's instructions. Briefly, 1/4 volume of ExoQuick solution was added to serums, and samples were incubated at 4 °C for 40 min. The mixture was centrifuged at 1,500 g for 30min, pelleted sEVs-enriched fraction was re-suspended in 100 μl particle-free PBS (Sigma, P4417). The supernatant was collected as sEVs-free serum. In some cases, serum sEVs, sEVs-free serum or whole serum were lysised in TRIzol (Life Tech, Carlsbad, CA) for RNA sample preparation.Total RNAs was extracted from serum sEVs and sEVs-free serums using TRIzol reagent according to the manufacturer's instructions. Small RNA libraries were generated using Illumina TruSeq Small RNA Sample kit (Illumina, San Diego, CA, RS-200-0012). Per manufacturer's recommendations, total RNA was subjected to sequential 3ʹ and 5ʹ adapter ligations (T4 RNA Ligase 2, Epicentre, LR2D1132K ) followed by reverse transcription (SuperScript II Reverse Transcriptase, Invitrogen, 18064-014) into a cDNA library. After PCR enrichment, the miRNA library was size (147nt and 157nt in length) selected from an 6% Novex TBE PAGE gel (Invitrogen, EC6265BOX) and the library quality was assessed on the Agilent Bioanalyzer 2100 system (Agilent).
ENA-FIRST-PUBLIC
2020-03-13T17:04:52Z
ENA-LAST-UPDATE
2019-09-25T15:13:31Z
External Id
SAMEA5974808
INSDC center name
Department of Pathology, Medical School of Nantong University, Nantong, China
INSDC first public
2020-03-13T17:04:52Z
INSDC last update
2019-09-25T15:13:31Z
INSDC status
public
Submitter Id
E-MTAB-8377:Sample 1
age
8
broker name
ArrayExpress
common name
house mouse
developmental stage
adult
fraction
serum exosome
genotype
wild type genotype
organism part
blood plasma
sample name
E-MTAB-8377:Sample 1
scientific_name
Mus musculus
sex
male
strain
C57BL/6