title: GSM3267678: 4KO(quad)-TM rep4; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted using the RNeasy kit (Qiagen, CA). Libraries were prepared using the Illumina (Illumina Inc. San Diego, CA, USA) TruSeq™ mRNA stranded kit as per manufacturer's instructions. Briefly, 500-1000 ng of total RNA was used for library preparation. ERCC (Ambion) synthetic spike-in 1 or 2 was added to each Total RNA sample and processed in parallel. The TruSeq method (high-throughput protocol) employs two rounds of poly-A based mRNA enrichment using oligo-dT magnetic beads followed by mRNA fragmentation (120-200 bp) using cations at high temperature. First and second strand cDNA synthesis was performed followed by end repair of the blunt cDNA ends. One single “A” base was added at the 3' end of the cDNA followed by ligation of barcoded adapter unique to each sample. The adapter-ligated libraries were then enriched using PCR amplification. The amplified library was validated using a DNA tape on the Agilent 4200 TapeStation and quantified using fluorescence based method. The libraries were normalized and pooled and clustered on the HiSeq3000/4000 flowcell on the Illumina cBot. The clustered flowcell was then sequenced on the Illumina HiSeq3000 system employing a single-end 101 cycles run.