GSM1422182: astrocyte_pre-sen_rep2; Homo sapiens; RNA-Seq
Total RNA was isolated using the RNeasy Mini Kit (Qiagen; Valencia, CA) according to the manufacturer’s instructions and the concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop; Rockland, DE) Libraries were prepared by selecting poly-A-containing mRNA from total cellular RNA using the Dynabeads mRNA direct kit (Life technologies). The mRNA was fragmented and a 5’phosphate was then added using T4 polynucleotide kinase (Life technologies). A 15% TBE-urea gel was used to size select the library for fragmented products of 60-200 nucleotides. The ends of the fragmented products were ligated with Illumina v1.5 RNA adapters using T4 RNA ligase 2 (New England Biolabs). Superscript II reverse transcriptase (Life technologies) was used to generate a cDNA library that was PCR-amplified using Phusion HiFi DNA polymerase (New England Biolabs). The cDNA library was purified on a 6% TBE gel (Life technologies). The quality of the library was verified prior to sequencing by cloning into Zero Blunt TOPO (Life technologies) and analyzing 6-10 clones by standard sequencing to determine the diversity of the library products, the abundance of rRNA, appropriate size of the products, and the presence of adapters. T he verified size-selected poly-A stranded cDNA library was submitted to the Next-Generation Sequencing Core (NGSC) University of Pennsylvania, Philadelphia PA for sequencing. The Illumina Solexa GAII platform was used to obtain 50-bp single-end sequencing reads, which were analyzed using the v1.5 pipeline.