GSM4647584: Basophil_Subject_10_CR_Day1; Homo sapiens; miRNA-Seq
Exosomes were isolated from the plasma of the study participants using Invitrogen's Total Exosome Precipitation Reagent (Thermo Fisher Scientific, Inc., Waltham, MA). miRNAs were then isolated from the exosomes using the QIAGEN miRNeasy Serum/Plasma Kit (QIAGEN Sciences, Germantown, MD). For basophils, peripheral blood samples were collected in EDTA tubes.  Human nucleated cells were separated from red blood cells using HetaSep reagent (Stemcell Technologies, Vancouver, Canada).  Basophils were isolated from nucleated cells using the Stemcell Technologies EasySep Human Basophil Enrichment Kit (isolation purity, mean ± SD, 94.0 ± 2.5%). Purified basophils were centrifuged at 1,500 rpm for 10 minutes. The pellet was resuspended in 200L of PBS, 1 mL of Trizol was added, and the purified basophils were stored at -80oC until RNA was isolated. Total RNA from basophils was isolated using the QIAGEN miRNeasy Micro extraction kit. The integrity of the total RNA samples was evaluated using a 2100 Bioanalyzer instrument (Agilent Technologies, Inc., Palo Alto, CA).  A Thermo Fisher Scientific NanoDrop™ 1000 Spectrophotometer was used to determine purity of RNA samples by measuring OD260/280 and OD260/230 ratios.  Only samples with RNA Integrity Number (RIN)s of ≥7 and OD260/280 and OD260/230 ratios >1.8 were used for further analysis.  Total RNA concentrations were determined using Thermo Fisher Scientific Qubit RNA HS Assay Kit. miRNA sequencing libraries prepared with the QIAseq miRNA Library Kit were sequenced using a NextSeq 500 instrument (Illumina, Inc., San Diego, CA). Libraries were quantified, adjusted to 4 nM each and pooled. The pooled libraries were further diluted down to 1.8 pM and loaded into a flow cell on an Illumina NextSeq 500. The sequencing specifications were 1x76 bases and 12 million targeted reads per sample.