GSM4006445: TET2_WT_LPS_310_TET2_LPS_RNA_Seq; Mus musculus; RNA-Seq
Mice were perfused with PBS and the midbrain was isolated and snap frozen. Midbrain tissue (25-50 mg) homogenized in TRIzol (Invitrogen) using a Precellys Lysing Kit (Bertin Technologies) with the MiniLys (Bertin). Total RNA was isolated and treated with DNase I (Qiagen), followed by a clean up using an RNeasy Mini Kit (Qiagen). The resulting RNA quantity was assessed by Nanodrop 8000 (Thermo Scientific) and quality was assessed with an Agilent RNA 6000 Nano Kit on a 2100 Bioanalyzer (Agilent Technologies). Libraries were prepared from 500 ng of total RNA using the KAPA RNA HyperPrep Kit with RiboseErase (v1.16) (Kapa Biosystems). RNA was sheared to 300-400 bp. Prior to PCR amplification, cDNA fragments were ligated to Bioo Scientific NEXTflex Adapters (Bioo Scientific). Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies), QuantiFluor® dsDNA System (Promega Corp), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems). Sequencing (single-end 75 bp) was performed on an Illumina NextSeq500 sequencer.