GSM2357189
GSM2357189: P15_1 ko; Mus musculus; RNA-Seq
Total RNA was Trizol extracted. RNA was treated with DNase (Qiagen, 79254) and purified on the RNeasy MinElute Cleanup Kit (Qiagen, 74204). 2000 ng of total RNA were treated with the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat; Illumina MRZH11124). Depleted RNA was precipitated 1 h at -80°C in three volumes of ethanol plus 1 µg of glycogen. RNA was then washed and resuspended in 36 µl of RNase free water. RNA fragmentation buffer (NEBNext® Magnesium RNA Fragmentation Module, E6150S) was added to the solution and the RNA was fragmented by incubation at 95°C for 3 minutes. For reverse transcription, cDNA first strand synthesis was performed with random examer primers. cDNA second strand synthesis was performed with dUNTPs, to ensure strand specificity. The RNA-seq library was synthetized with KAPA Hyper prep kit (KK8504), a treatment with USER enzyme (NEB, M5505L) was added to digest the unspecific strand. The libraries were pooled (4/lane) and sequenced on the illumina HiSeq, which yielded approximately 40 million single-end reads per sample.
GEO
GSM2357189
GEO Accession
GSM2357189