GSM5655484: C5_KO_Mac; Mus musculus; RNA-Seq
Total RNA was isolated using QIAzol (Qiagen) and chloroform extraction followed by purification on Direct-zol RNA miniprep kit (Zymo Research) and then quantified using Nanodrop 2000 UV-visible spectrometry RNA sequencing libraries were generated using SENSE mRNA-Seq library prep kit according to manufacturer's instructions (Lexogen GmbH, Vienna, Austria). In brief, up to 200 ng of total RNA was prepared and incubated with magnetic beads coated with oligo-dT, then all other RNAs except mRNA were removed by washing. Library preparation was then initiated by random hybridization of starter/stopper heterodimers to the poly(A) RNA still bound to the magnetic beads. These starter/stopper heterodimers contain Illumina-compatible linker sequences. A single-tube reverse transcription and ligation reaction extends the starter to the next hybridized heterodimer, where the newly synthesized cDNA insert was ligated to the stopper. Second-strand synthesis was performed to release the library from the beads. The resulting double-stranded library was purified and amplified (15 PCR cycles) after adding the adaptors and indexes. Finally, libraries were quantitated by qPCR using KAPA Library Quantification Kit (Kapa Biosystems, Inc., MA, USA) and validated using Agilent High Sensitivity DNA Kit on a Bioanalyzer (Agilent Technologies, CA, USA)