title: GSM4408532: EV_644; Homo sapiens; miRNA-Seq
library_construction_protocol: Breast milk samples were centrifuged at 200g for 10 mins to remove fat globules, then 3,000g for 15 mins to pellet cellular debris. Extracellular vesicle (EV) RNA was isolated with the ExoEasy Maxi Kit and the miRNeasy RNA Isolation Kit (Qiagen) according to manufacturer instructions. Libraries were constructed with the HTG EdgeSeq WT-miRNA Assay (Tucson, Arizona). Samples were processed by adding HTG Lysis Buffer to lyse and/or permeabilize the RNA available to subsequently bind to corresponding target-specific Nuclease Protection Probes (NPPs). The lysed samples was transferred to a 96-well micro-titer plate. Target capture was done by adding NPPs to the lysed samples to hybridize to the target mRNA. Then S1 nuclease was added to digest non-hybridized miRNA and excess NPPs, leaving target mRNA/NPP duplexes. S1 digestion was terminated by heat denaturation of S1 enzyme. The processed samples were used as template to set up PCR reactions with primer tags. These tags share common sequences that are complementary to 5'-end and 3'-end “wing” sequences of the probes and common adaptors required for cluster generation for deep sequencing. The library was prepared using HTG EdgeSeq PCR processing. Following library preparation, it was quantified using HTG EdgeSeq KAPA Library Quantification for Illumina Sequencing. All samples and controls were quantified in triplicate. Targeted miRNA-Seq
attributes.tag: GEO Accession
attributes.value: GSM4408532