title: GSM3768804: I12F-BNST; Rattus norvegicus; miRNA-Seq
library_construction_protocol: The anterodorsal BNST (adBNST) was punched from frozen brains, total RNA was extracted using Trizol reagent (Ambion, Life Technologies) and was treated with a DNA-free kit (Ambion, Life Technologies). Total RNA (215ng) was used for the library construction as described below. MiRNA Libraries were prepared using the Bioo Scientific NEXTflex Small RNA-Seq kit v3. Total RNA (215ng) was denatured and NEXTflex adapters were ligated to the 3' and 5' ends of the RNA. After adapter ligations and cleanups, the adapter-ligated RNA entered reverse transcription, resulting in a cDNA first strand synthesis product. The reverse transcription products were then isolated and amplified by PCR (22 cycles). Successful library production and isolation was confirmed by the Agilent High-Sensitivity Screentape assay. Quantification of libraries was performed using Qubit Picogreen and KAPA quantitative real-time polymerase chain reaction (qRT-PCR) assays. Libraries were normalized, pooled, denatured and sequenced on the HiSeq 2500 using Rapid v2 sequencing reagents and flowcell. All samples were processed using the small RNA-seq pipeline implemented in the bcbio-nextgen project (https://bcbio-nextgen.readthedocs.org/en/latest/).
attributes.tag: GEO Accession
attributes.value: GSM3768804