title: GSM2806218: 6-14A.RNA.Young; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated from 100 mg brain tissue by TRIzol solubilization and phenol-chloroform extraction followed by ethanol precipitation.  Purified RNA was subsequently treated with RNase-free DNase (Qiagen). RNA sequencing libraries were prepared as previously described with a few changes, such as omission of the polyA+ selection step and addition of a double-stranded nuclease (DSN)-mediated step to deplete highly abundant RNA species, such as ribosomal RNA. Briefly, purified RNA was fragmented using RNA Fragmentation Reagent (Thermo Fisher) for 5 min at 70°C and followed by addition of a 5’ phosphate by treating with T4 polynucleotide kinase (NEB). The RNA fragments were then size-selected to 60-250 bp on a 15% TBE-urea gel. Illumina v1.5 RNA adapters were ligated to the 3’ and 5’ ends of the fragmented products using T4 RNA ligase 2, truncated (NEB) and T4 RNA ligase (Promega), respectively. cDNA libraries were generated using Superscript II (Invitrogen) and PCR-amplified using Phusion high-fidelity DNA polymerase (NEB). Libraries were purified on a 6% TBE gel (Invitrogen), treated with DSN and PCR re-amplified. Quality of the resulting sequencing libraries were verified by cloning into Zero Blunt TOPO (Invitrogen) and analyzing 6-10 clones by standard Sanger sequencing to determine the presence of adapters, appropriate size selection, abundance of ribosomal RNA, and the diversity of the library products. The libraries were pooled and sequenced on an Illumina NextSeq 500 using the standard protocols for 75 bp single-read sequencing.
attributes.tag: GEO Accession
attributes.value: GSM2806218