GSM3595698: microRNA NGS: hDF2 GFP; Homo sapiens; miRNA-Seq
Cells were washed with phosphate-buffered saline (PBS), (pH 7.4) and lysed using RNA Blue reagent (Top-Bio). After 1-bromo-3-chloropropane (Sigma-Aldrich) addition and centrifugation (21.000 × g, 15 minutes, 4 °C), the cell lysates were separated into three layers. The top aqueous layer containing RNA was transferred to a new tube and precipitated by addition of isopropanol, followed by 10 minutes incubation on ice and centrifugation (18.000 × g, 10 minutes, 4 °C). RNA pellets were washed with 70% ethanol (8.500 × g, 10 minutes, 4 °C), air-dried, solubilized in PCR grade water (Top-Bio), and RNA integrity was analysed (RIN>8). NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set1, 2; New England Biolabs; Ipswich, MA, USA) was used to prepare libraries for further sequencing according to the manufacturer's instructions. Briefly, 800 ng of total RNA were used to create size selected small RNA library. The sequencing was performed with 2.0 pM library using the NextSeq® 500/550 High Output Kit v2 (75 cycles; Illumina; San Diego, CA, USA).
GEO Accession
GSM3595698