title: GSM8455344: HPC215.counts; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated from different brain regions using the ALLPrep isolation kit (Qiagen). Before extraction, frozen tissues were transferred to a 2 ml lysing tube containing 1.4 mm zirconium oxide beads (Precellys) and 600 μl of RLT buffer (provided by the Kit). Samples were homogenized using FastPrep (20 seconds, 5000 rpm) and centrifuged at full speed for 5 minutes. The supernatant was transferred to an ALLPrep spin column and isolation proceeded according to the manufacturer's protocol. To improve the quality of the RNA, b-mercapto-ethanol was added to the RLT buffer at the recommended concentration and samples were further treated with DNaseI to digest contaminating DNA. RNA quality and concentration were measured using Agilent Bioanalyser (Agilent Technologies) or Nanodrop. After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. For prokaryotic samples, rRNA is removed using the Ribo-Zero kit that leaves the mRNA. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment.  The quality control of library consists of three steps: (1) Qubit 2.0: tests the library concentration preliminarily. (2) Agilent 2100: tests the insert size. (3) Q-PCR: quantifies the library effective concentration precisely.