title: GSM5171786: GABAergic CB1-KO - open field - rep 4 [2015_01_47_dlx_ko_OF_613]; Mus musculus; RNA-Seq
library_construction_protocol: In order to microdissect only the Cornus Ammonis from the hippocampus, we first removed the dentate gyrus (DG) from the rest of the hippocampus according to the protocol of Hideo Hagihara (Hagihara et al., 2009). The remaining hippocampal tissue was then dissected out in one-piece and snap-frozen on dry ice. In order to sustain the amount and quality of the hippocampal RNA for sequencing, the extraction and purification of RNA was performed as previously described (Lerner et al., 2018). To summarize, we followed the instructions from the RNeasy Mini kit (Qiagen) with a slight modification during the homogenization step. Frozen tissue was homogenized in 600μLRLT buffer with 1% β-mercaptoethanol (according to the manufacturer ́s protocol), though with the addition of 200 μL chloroform. Apart from this step, therewere no other changes made. Obtained RNA samples were treated with DNase I to degrade any possible contamination by genomic DNA. Samples were then eluted in 30 μL RNase-free water. The working bench and the tools used were cleaned before and during the extraction with RNase away plus (MßP, San 128Diego, CA, USA) to avoid any RNA degradation. The hippocampal transcriptome sequencing occurred at the Core Facility Genomics (Institute of Molecular Biology gGmbH, IMB; Mainz). Next generation sequencing (NGS) library preparation was performed in two steps. First, cDNA was generated using NuGEN ́s Ovation RNA-seq system v2, from an input amount of 10 ng of total RNA, following the kit ́s instructions from the year 2012 (NuGEN, The Netherlands). Samples were amplified using the single primer isothermal amplification (SPIA) method from NuGEN. The resulting purified cDNA was quantified using the Qubit dsDNA HS assay kit in a Qubit 2.0 Fluorometer (Life 141Technologies, Germany). Afterward, the cDNA was profiled on a high sensitivity DNA chip using a 2100 Bioanalyzer (Agilent Technologies, Germany) as quality control. From the total cDNA, 1.5 μg were fragmented using a Covaris S2 focused-ultrasonicator (Covaris, UK), with the following parameters: 1) Duty cycle = 10%; 2) Intensity = 5; 3) Cycles/Burst = 200; 4) Time 145= 160 seconds; and 5) Water level = 15. After the fragmentation, the resulting material was once again quantified and profiled using a Qubit 2.0 and a 2100 Bioanalyzer, respectively, as described above. This process was repeated as an extra quality control point to ensure an optimal fragmentation. Secondly, NGS libraries were generated from 100 ng of fragmented cDNA usingNuGEN ́s Ovation ultralow system v2, following the kit ́s manual from the year 2014 (NuGEN, The 151Netherlands). Libraries were amplified in 7 PCR cycles and purified using beads. These purified libraries were quantified as described above, and profiled on a DNA 1000 Chip using a 2100 Bioanalyzer as the last quality control. Libraries representing all the experimental groups were pooled into individual pools containing 12 libraries, all of them in equimolar ratio. Each pool was loaded into four lanes of an Illumina's HiSeq flowcell and ran on a HiSeq 2500 in High-output mode (Illumina, USA), generating single-reads 50 base pairs long with an average yield of 52 million reads per library.