GSM4709108: Tfap2a rep3; Mus musculus; RNA-Seq
RNA was isolated from 20-80mg of mouse brain tissue from B6N, Tfap2a+/- and Tfap2b+/- (stored in RNAlater) following the Qiagen RNeasy protocol. First, the tissue samples were homogenized with the TissueLyser (Qiagen) at 25Hz for 2 * 2 minutes in Qiazol lysis buffer. 140µl chloroform was added and after 15min centrifugation at 12,000g the aqueous phase containing the RNA was extracted. 1.5 volumes of ethanol were added and the samples were washed with Qiagen's RWT buffer on a Qiagen RNeasy spin column. RNA was treated with 10µl of DNase I on column for 15min followed by a wash with RWT. After further washes with RPE buffer, the purified RNA was eluted with 50µl water yielding between 5 and 22µg RNA. After quality control using Agilent's Bioanalyzer, sequencing libraries were prepared from 500ng of total RNA per sample following Roche's stranded “KAPA RNA HyperPrep” library preparation protocol for single indexed Illumina libraries: First the polyA-RNA fraction was enriched using oligo-dT-probed paramagnetic beads. Enriched RNA was heat-fragmented and subjected to first strand synthesis using random priming. The second strand was synthesized incorporating dUTP instead of dTTP to preserve strand information. After A-tailing, Illumina sequencing compatible adapters were ligated. Following bead-based clean-up steps the libraries were amplified using 11 cycles of PCR. Library quality and size was checked with qBit, Agilent Bioanalyzer and qPCR. Sequencing was carried out on an Illumina HiSeq 4000 system in PE75bp mode yielding between 27 and 37 million fragments per sample.
GEO Accession
GSM4709108