Multina electrophoresis device

Brain tissue from five TBI and five sham-operated rats was used for methyl-binding domain sequencing (MBD-seq) and RNA-sequencing (RNA-seq). DNA and RNA were co-purified from the perilesional cortex, ipsilateral hippocampus, or ipsilateral thalamus using a DNeasy Blood&Tissue kit (#69504, Qiagen, Hilden, Germany). Quality control of the total RNA was performed using a MultiNA electrophoresis device (Shimazu, Kyoto, Japan).

The mRNA library preparation and RNA-sequencing were performed as described in Lipponen et al. [69 (link)]. Briefly, mRNA was enriched using Dynabeads Oligo (dT)25 beads (#61002, Invitrogen, Carlsbad, CA, USA), and the sequencing libraries were compiled with the NEBNext mRNA Library Prep Reagent Set (#E6100S, New England Biolabs, Ipswich, MA, USA). Quality control of the sequencing libraries was performed with a MultiNA electrophoresis device (Shimazu, Kyoto, Japan). Sequencing of the mRNA libraries for the perilesional cortex and hippocampus was carried out with an Illumina Genome Analyzer IIx (San Diego, CA, USA), and for the thalamus using an Illumina HiSeq 2000 (San Diego, CA, USA). The Illumina Off-Line Basecaller v1.8 was used for base-calling. RNA-seq raw data can be downloaded from the NCBI Gene Expression Omnibus (GEO; series accession number GSE80174).

RNA was extracted from the perilesional cortex, thalamus, and hippocampus DNaesy Blood & Tissue kit (#69504, Qiagen, Hilden, Germany) followed by DNase digestion. mRNA from 2 μg of total RNA was enriched using Dynabeads Oligo (dT)₂₅ beads (#61002, Invitrogen, Carlsbad, CA, USA). The sequencing library was prepared with the NEBNext mRNA Library Prep Reagent Set (#E6100S, New England Biolabs, Ipswich, MA, USA). Quality control of the total RNA and sequencing libraries was performed using a MultiNA electrophoresis device (Shimazu, Kyoto, Japan). Then, sequencing of the mRNA library (RNA-Seq) for the perilesional cortex and hippocampus was carried out with an Illumina Genome Analyzer IIx (San Diego, CA, USA) with 36 cycles, and for the thalamus using Illumina HiSeq 2000 (San Diego, CA, USA) with 50 cycles. Base-calling was performed using an Illumina Off-Line Basecaller v1.8. RNA-Seq raw data were saved to the NCBI Gene Expression Omnibus (GEO; series accession number GSE80174).