GSM4649008: R099-H-001; Mus musculus; RNA-Seq
Cells were resuspended in RLT Plus buffer (Qiagen) and stored at -80 degrees. RNA extraction was carried out using a RNAeasy Plus micro kit (Qiagen) following the manufacturers instructions. Total RNA quality and quantity was assessed using Agilent 2100 Bioanalyser and a RNA Nano 6000 kit (Agilent Technologies). 30-260ng of Total RNA with a RIN value >8 was depleted of ribosomal RNA using the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat), (New England BioLabs, NEB) Sequencing libraries were prepared using the NEB® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB). The steps included RNA fragmentation and priming, 1st strand cDNA synthesis, 2nd strand cDNA synthesis, adenylation of 3' ends, adapter ligation, PCR amplification (11-cycles) and validation. The manufacturer's instructions were followed. The libraries were validated using the Agilent 2100 Bioanalyser and a high-sensitivity kit (Agilent Technologies) to ascertain the insert size, and the Qubit® (Life Technologies) was used to perform the fluorometric quantitation. Following validation, the libraries were normalized to 10nM, pooled together and clustered on the cBot™2 following the manufacturer's recommendations. The pool was then sequenced using a 75-base paired-end (2x75bp PE) dual index read format on the Illumina® HiSeq4000 according to the manufacturer's instructions.
GEO Accession
GSM4649008