title: GSM8421613: Y28_NesGrnOE-koBG_7891; Mus musculus; RNA-Seq
library_construction_protocol: The brain was removed, cerebellum, olfactory bulb nad brainstem removed, cut sagittal, one half for RNAseq was immediately frozen in liquid nitrogen and stored at -80° before RNA extraction. Total RNA was isolated using RNeasy plus micro kit (Qiagen). Quantity of total RNA was assessed with Qubit 2.0 and quality was checked using a RNA 6000 Nano chip on Agilent's bioanalyzer. 200 ng of total RNA was send to Novogene (Cambridge, UK) for library preparation and sequencing. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. This method is known as polyA-tailed mRNA enrichment. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The workflow of library construction was as follows: mRNA enrichment, fragmentation, reverse transcription, second strand cDNA synthesis, end repair & A-tailing, adapter ligation, size selection, PCR amplification. Barcoded mRNA seq libraries were prepared from 200 ng total RNA.  mRNA was randomly fragmented and cDNA synthesis proceeded through the use of random hexamers and the reverse transcriptase enzyme. Once the synthesis of the first chain was finished, the second chain was synthesized with the addition of an Illumina buffer. With this and together with the presence of dNTPs, RNase H and polymerase I from E.coli, the second chain was obtained by Nick translation. The resulting products went through purification, end-repair, A-tailing and adapter ligation. Fragments of the appropriate size were enriched by PCR, where indexed P5 and P7 primers were introduced, and final products were purified. The library was checked with Qubit 2.0 and real-time PCR for quantification and bioanalyzer Agilent 2100 for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms on Illumina Novaseq 6000 platform, according to effective library concentration and data amount. Sequencing strategy was paired-end 150 cycles (PE150).