GSM8616486: IB25; Homo sapiens; RNA-Seq
Snap-frozen mouse kidney tissue (<100 mg) was homogenized in 800 µl of Tri Reagent using a Minilys tissue homogenizer at the highest speed for 45 seconds and then incubated for 5 minutes. Total RNA was extracted from 600 µl of the supernatant using the Direct-zol RNA miniprep Plus (Zymo Research), including on-column DNase I digestion. The RNA was eluted in 100 µl of water and subjected to mRNA polyA enrichment using the Dynabeads mRNA DIRECT Micro kit (Thermo Fisher 61021). After two rounds of washing and enrichment of mRNA following the manufacture's protocol, polyA+ mRNA was eluted in 10 µl of water. We proceeded with ~50 ng of polyA+ mRNA as the starting material. Reverse transcription and strand-switching was done using Maxima H Minus Reverse Transcriptase following the Nanopore protocol (SQK-DCS109). After end-prep, adapter ligation, and AMPure XP bead binding, sequencing was conducted on R9.4.1 flow cells using the GridIon platform.