GSM3504961: 15_8317TrPos_Pos; Mus musculus; RNA-Seq
RNA was extracted from using the miRNeasy micro kit (Qiagen) and quantified as previously descirbed (Seumois G et al. Am J Clin Exp Immunol. 2012; Seumois G et al, Nat Immunol. 2014) 5 ng of purified total RNA was used for poly(A) mRNA selection (Picelli S et al, Nat Protoc. 2014). Purified total RNA (5 ng) was amplified following the Smart-seq2 protocol and amplified cDNA purified with AMPure XP beads (1:1 ratio; Beckman Coulter). 1 ng of cDNA was used for library preparation using Nextera XT DNA sample preparation kit and index kit (Illumina, catalog# FC-131-1096). Samples were sequenced using a HiSeq2500 (Illumina) to obtain 50-bp single end reads. To reduce assay-to-assay variability, both whole transcriptome amplification and sequencing library preparations were performed in a 96-well format. Quality control steps were included to determine total RNA quality and quantity, the optimal number of PCR pre-amplification cycles, and fragment size selection (Bioanalyzer, Agilent), samples that failed quality control were eliminated from further downstream steps. Barcoded Illumina sequencing libraries were generated utilizing an ancillary automated platform (Biomek Fxp, Beckman Coulter). Libraries were sequenced on the HiSeq2500 Illumina platform to obtain 50-bp single-end reads (HiSeq SBS and SR Cluster v4 kits, Illumine catalog# FC-401-4002 and GD-401-4001, respectively), generating an average of 11.4 million mapped reads / sample.
GEO Accession
GSM3504961