title: GSM8454410: H2axfl/flCre- + IL-1β, rep1; Mus musculus; RNA-Seq
library_construction_protocol: After sorting, neuronal nuclei were centrifuged at 1000 g for 7 min at 4°C and pellet was resuspended in RLT buffer (Qiagen) supplemented with 1% (vol/vol) β-mercaptoethanol (final concentration 143mM, Sigma). Samples were homogenized through QiaShredder columns (Qiagen) and stored at -80°C until processing. Bulk nuclear RNA extraction was performed using RNeasy Micro Kit (Qiagen) following manufacturer's instructions. RNA concentration was quantified using Qubit RNA HS Assay Kit on a Qubit Fluorometer (Invitrogen). The quality was evaluated on an Agilent 4150 TapeStation with the High Sensibility RNA ScreenTape kit (Agilent). Libraries were then produced using the llumina® Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit (Illumina), according to the manufacturer's instructions, including a few modifications. Ribosomal RNA (rRNA) and mitochondrial ribosomal RNA were depleted using DNA probes that bind to rRNA targets, both of which are enzymatically digested. The remaining RNAs were fragmented for 4 min and reverse transcribed into cDNA, retaining the strand orientation information. The fragments were indexed and amplified by PCR. The libraries were quantified using the Qubit 1X dsDNA HS kit on the Qubit Fluorometer (Invitrogen), and evaluated on an Agilent 4150 TapeStation with the High Sensitivity D5000 Kit (Agilent).