GSM3476491: Urine sEV - 39yo female; Homo sapiens; ncRNA-Seq
Total RNA was extracted from sEV isolates using the miRNeasy Micro kit (Qiagen, Valencia, CA) according to the manufacturer's suggested protocol NEBNext small RNA sample library preparation kit (New England BioLabs, Ipswich, MA) was used with ~5 ng of total RNA determined by Bioanalyzer RNA 6000 Pico Kit (Agilent, Santa Clara, CA) in a 5 µL solution as input, following the manufacturer's suggested protocol excepting a modification of library size selection to increase small RNA detection sensitivity and specificity. After 15 cycles of PCR for indexing and library enrichment, an equal-volume of 10 ml PCR mix (library without size selection) per sample together with the same volume of the negative control were pooled, followed by DNA cleanup using DNA Clean & Concentrator (Zymo Research, Irvine, CA) and mixed with 135 and 319 bp custom-made ladders targeting the library 16 – 200 nt small RNA cDNA insert. Next, precise size selection of the 135-319 bp library via 2.75% agarose gel electrophoresis was performed, library concentration was measured by qPCR using the NEBNext Library Quant Kit (New England BioLabs) on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific).
GEO Accession
GSM3476491