GSM6939585: CA1-tissue, wildtype [C57BL/6], ECL, biol rep 1; Mus musculus; RNA-Seq
RNA library preparations for transcriptome analysis were performed using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (New England Biolabs, #E6420) according to the manufacturer's instructions. Briefly, isolation of the dentate gyrus from individual tissue cultures was performed using a scalpel. One isolated dentate gyrus was transferred to 7.5 µl lysis buffer (supplemented with murine RNase inhibitor) and homogenized using a pestill. Samples were centrifuged for 30 seconds at 10000g and 5 µl of supernatant were collected from individual samples and further processed. After cDNA synthesis, cDNA amplification was performed according to the manufacturer's protocol with 12 PCR cycles. The cDNA yield was subsequently analyzed by a High Sensitivity DNA assay on a Bioanalyzer instrument (Agilent). The amount of cDNA was adjusted to 10 ng for further downstream applications. After fragmentation and adaptor ligation, dual index primers (New England Biolabs, #E7600S) were ligated in a library amplification step using 10 PCR cycles. Libraries were finally cleaned up with 0.8X SPRI beads following a standard bead purification protocol. Library purity and size distribution were assessed with a High Sensitivity DNA assay on a Bioanalyzer instrument (Agilent). We quantified the libraries using the NEBNext Library Quant Kit for Illumina (New England Biolabs, #E7630) based on the mean insert size provided by the Bioanalyzer. A 10 nM sequencing pool (120 µl in Tris-HCl, pH 8.5) was generated for sequencing on the NovaSeq6000 sequencing platform (Illumina; service provided by CeGaT GmbH, Tübingen, Germany). We performed a paired-end sequencing with 150 bp read length.