GSM3502325
GSM3502325: KOWT_WT1_cell; Mus musculus; RNA-Seq
Cerebral cortex 7-10 weeks of age was dissected on ice in phosphate buffer saline. The tissue was flash-frozen in liquid nitrogen and stored at -80°C. Total RNA was extracted from 1/16th of a whole cortex using RLT buffer following RNeasy Micro Kit (Qiagen). Nuclear RNA was isolated following a modified version of the protocol described by Mo et al., 2015. Briefly, half of a cortex was homogenized in 0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH using a glass dounce homogenizer. Nuclei were isolated via centrifugation at 10,000g for 18 minutes at 4°C (Sorvall HB-4) by pelleting through a 30% iodixanol density gradient (Sigma D1556). RNA was isolated from nuclei by resuspending pellet in RLT buffer following the RNeasy Micro Kit (Qiagen). RNA libraries were generated from 250ng total and nuclear RNA with NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB) using a modified amplification protocol (37°C, 15 minutes; 98°C, 30 seconds; (98°C, 10 seconds; 65°C, 30 seconds; 72°C, 30 seconds)x13; 72°C, 5 minutes; 4°C hold. RNA libraries were pooled at a final concentration of 8-10nM RNA was sequenced using Illumina HiSeq 2500 or 3000 with the Genome Technology Access Center (GTAC) at Washington University in St. Louis, typically yielding 20-30 million single-end reads per sample.
GEO
GSM3502325
GEO Accession
GSM3502325