title: GSM1686400: Wildtype 4; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted as previously described with slight modification (Fertuzinhos S, Li M, Kawasawa YI, Ivic V, Franjic D, Singh D, Crair M, and Sestan N. Laminar and temporal expression dynamics of coding and noncoding RNAs in the mouse neocortex. Cell Rep 6: 938-950, 2014). Briefly, frozen muscle tissue was pulverized in liquid nitrogen using mortar and pestle, followed by bead mill homogenization (Bullet Blender, Next Advance) using stainless steel beads (Next Advance, cat# SSB14B) and mirVana RNA isolation kit (Life Technologies). Optical density values of extracted RNA were measured using NanoDrop (Thermo Scientific) to confirm an A260:A280 ratio above 1.9. RIN was confirmed as greater 7 for each sample using Bioanalyzer RNA 6000 Nano Kit (Agilent Technologies). The cDNA libraries were prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina) as per the manufacturer’s instructions. The final product was assessed for its size distribution using Bioanalyzer DNA High Sensitivity Kit (Agilent Technologies) and for its concentration using Kapa library quantification kit (Kapa Biosystems). Eight libraries were pooled per HiSeq lane, followed by on-board cluster generation on a Rapid Run single-end flow cell and subsequent 50 cycles sequencing (v3 sequencing kit) according to the manufacturer's instructions (HiSeq 2500, Illumina). Demultiplexed and quality filtered mRNA-seq reads were then aligned to mouse reference assembly (mm10) using TopHat (v.2.0.9). The uniquely mapped reads were used to calculate the normalized expression level of genes, as fragments per kilobase of exon per million fragments mapped (FPKM), using Cufflinks (v.2.0.2). The cDNA libraries were prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina) as per the manufacturer’s instructions. The final product was assessed for its size distribution using Bioanalyzer DNA High Sensitivity Kit (Agilent Technologies) and for its concentration using Kapa library quantification kit (Kapa Biosystems). Six libraries were pooled per HiSeq lane, followed by on-board cluster generation on a Rapid Run single-end flow cell and subsequent 50 cycles sequencing (v3 sequencing kit) according to the manufacturer's instructions (HiSeq 2500, Illumina).
attributes.tag: GEO Accession
attributes.value: GSM1686400