Rneasy plus minispin column kit

RNA extraction was performed using the Qiagen RNeasy Plus minispin column kit, eluting in 50 µL of RNase-free water, and using an additional DNase step. RNA concentration was determined using the Qubit RNA High-Sensitivity kit (Life Technologies). Reverse transcription was performed using the Invitrogen SuperScript III kit according to the manufacturer's instructions. TaqMan qPCR and TaqMan LDA card assays were performed using TaqMan Universal PCR Master Mix and assays (Applied Biosystems) according to the manufacturer's guidelines. Results were normalized to the housekeeping gene Gapdh and analyzed with HTqPCR (Dvinge and Bertone 2009 (link)). The following TaqMan assays were used: mGapdh (Mm99999915_g1), mFoxG1 (Mm02059886_s1), mFoxo3 (Mm01185722_m1), mGfap (Mm01253033_m1), mAqp4 (Mm00802131_m1), mS100b (Mm00485897_m1), mNestin (Mm00450205_m1), mOlig2 (Mm01210556_m1), mBlbp (Fabp7) (Mm00445225_m1), mSox2 (Mm03053810_s1), mDnmt1 (Mm01151063_m1), mDnmt3b (Mm01240113_m1), mTet3 (Mm00805756_m1), and hGAPDH (Hs02758991_g1).

RNA extraction was performed using the Qiagen RNeasy Plus minispin column kit, eluting in 50 µL of RNase-free water, and using an additional DNase step. RNA concentration was determined using the Qubit RNA High-Sensitivity kit (Life Technologies). Sample concentration and quality was assessed using the Aligent Tapestation and Nanodrop, only samples with a RIN value of 9.6 or above were used. For RNA-seq we used RNA-Seq Quantification Library (Low input Library) BGISEQ-500RS. Fastq files were trimmed via Trimgalore (version 0.5.0) and aligned with Kallisto (version 0.44.0) to the mm10 transcriptome (Martin, 2011; Bray et al., 2016). Tximport (version 1.8.0) was used to summarize read counts before subsequent normalization and differential expression as per DESeq2 (version 1.27.32). All downstream analysis and visualization were completed following regularized logarithm transformation (rlog) via DESeq2. GO enrichment analysis was completed via the hypergeometric test using the R package clusterprofiler (version 3.15.4) on differentially expressed genes (> 1.5 log₂ fold change, < 0.05 FDR) (Yu et al., 2012).