GSM4159672: RNA_T78_SD_T78S2; Mus musculus; RNA-Seq
Mice were anesthetized with isoflurane prior to decapitation. Cortex was rapidly dissected and flash frozen in liquid nitrogen. Frozen cortex of each individual was ground in liquid nitrogen and stored at -80°C until further use. Tissue from each mouse was distributed to the two protocols (RNAseq and ATAC-seq), such that both datasets originate from the exact same set of individuals. Total RNA was extracted using the miRNeasy kit (Qiagen; Hilden, Germany) following the manufacturer's instructions. RNA-seq libraries were prepared using 1000 ng of total RNA and the Illumina TruSeq Stranded mRNA reagents (Illumina; San Diego, CA, USA) on a Sciclone liquid handling robot (PerkinElmer; Waltham, MA, USA) using a PerkinElmerdeveloped automated script. Libraries were sequenced on the Illumina HiSeq 2500 sequencer, producing >36 million (median 55 million) mappable single-end 100 bp reads. ATAC-seq was performed as followed, 100'000 nuclei were treated with 2.5 μl Tagment DNA enzyme (Nextera DNA Sample Preparation Kit, Illumina) in transposition buffer (10mM Tris Base, 5mM MgCl2, 10% DMSO, pH 7.6) at 37°C for 30 minutes, followed by cleanup on a Qiagen Minelute column. Fragments >1kb in size were removed using 0.6X, then 1X, volumes of AmpureXP beads (Beckman Coulter Life Sciences; Indianapolis, IN, USA). DNA fragments were subjected to 11 cycles of PCR amplification with Nextera dual index primers (Illumina) and the NEBNext High Fidelity 2X PCR Master Mix (New England Biolabs; Ipswich, MA, USA). PCR reactions were cleaned up with one volume AmpureXP beads, quantified by Qubit (ThermoFisher Scientific; Waltham, MA, USA) and quality controlled by Fragment Analyzer (Advanced Analytical Technologies; Ankeny, IA, USA). Libraries were sequenced on the Illumina HiSeq 2500 sequencer, producing >25 million (median 41 million) mappable 50 bp paired-end reads per sample after removal of duplicate and mitochondrial sequences.
GEO Accession
GSM4159672