Name: GSM6376808
Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Surgically removed brain tissue was immediately placed in a sterile pre-labelled container and subsequently stored in -80_C freezer until analyzed. Half of the tissue was put in a routinely used fixative, 4% buffered formalin (Histo- Lab Products AB, Gothenburg, Sweden, catalogue no 02176). The samples were fixed for 24–72 h and then paraffin-embedded and processed by hardware Tissue tek VIP (Sakura, CA, USA). From the fresh frozen contused brain tissue, samples of ca 5mm2 were taken for scRNA sequencing. Single nuclei RNAseq: Protocol based on published nuclei isolation protocol (Södersten et al., 2018). Around 20 μg of -80 C-conserved tissue were thawed and dissociated in ice-cold lysis buffer (0.32M sucrose, 5 mM CaCl2, 3 mM MgAc, 0.1 mM Na2EDTA, 10 mM Tris-HCl pH 8.0, 1 mM DTT) using a 1 ml glass douncer (Wheaton). The homogenate was slowly and carefully layered in the centrifuge tubes on top of a sucrose layer (1.8 M sucrose, 3 mM MgAc, 10 mM Tris-HCl pH 8.0, 1 mM DTT) to create a gradient, and then centrifuged at 15500 rpm for 2 h 15 min. Following centrifugation, the supernatant was removed and the pellet softened for 10 minutes in 100 μl of nuclear storage buffer (15% sucrose, 10 mM Tris-HCl pH 7.2, 70 mM KCl, 2 mM MgCl2) prior resuspension in 300 μl of dilution buffer (10 mM Tris-HCl pH 7.2, 70 mM KCl, 2 mM MgCl2, Draq7 1:1000). The suspension was then filtered (70 μm cell strainer) and sorted via FACS (FACS Aria III, BD Biosciences) at 4° C with low flowrate, using a 100 μm nozzle (Pipette tips and Eppendorf tubes for transferring nuclei were pre-coated with 1% BSA). Bulk RNAseq: Total RNA was isolated from nuclei using the RNeasy Mini Kit (Qiagen). Single nuclei RNAseq: 8500 nuclei were sorted for single-nuclei RNA-sequencing and then loaded onto the Chromium Next GEM Single Cell 3' Kit (10x Genomics). Sequencing libraries samples were multiplexed and sequenced on a Novaseq machine using a 150-cycle kit using the recommended read length from 10x Genomics. Bulk RNAseq: Libraries were generated using Illumina TruSeq Stranded mRNA library prep kit (poly-A selection) and were sequenced on an Illumina NextSeq500 machine (paired-end 2×150bp).