GSM7039135: TBI_HuBrainCTL_Nuclei_529F_bulk_S9; Homo sapiens; RNA-Seq
The nuclei isolation from the brain tissue was performed according to Jönsson et al., 2021. In brief, the tissue was thawed and dissociated in ice-cold lysis buffer (0.32 M 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 tissue douncer (Wheaton). The homogenate was carefully layered on top of a sucrose cushion (1.8 M sucrose, 3 mM MgAc, 10 mM Tris-HCl, pH 8.0, and 1 mM DTT) before centrifugation at 30,000 × g for 2 hours and 15 min. Pelleted nuclei were softened for 10 min in 100 ml of nuclear storage buffer (15% sucrose, 10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) before being resuspended in 300 ml of dilution buffer (10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) and run through a cell strainer (70 mm). Cells were run through the FACS (FACS Aria, BD Biosciences) at 4°C at a low flow rate using a 100 mm nozzle (reanalysis showed >99% purity). Single cell/nuclei: Nuclei/cells were directly loaded onto the Chromium Next GEM Chip G or Chromium Next GEM Chip K Single Cell Kit along with the reverse transcription mastermix following the manufacturer's protocol for the Chromium Next GEM Single Cell 5' Kit (10x Genomics, PN-1000263) to generate single-cell gel beads in emulsion. cDNA amplification was done as per the guidelines from 10x Genomics using 15 cycles of amplification. Bulk RNAseq (truseq): Total RNA was isolated from nuclei using the RNeasy Mini Kit (Qiagen). Bulk RNAseq (pacbio)Total RNA was obtained from tissue samples using miRNA Easy Mini Kit (Qiagen). RNA samples were subsequently put on dry ice and shipped to the National Genomics Infrastructure of Sweden. There, input QC of samples was performed on the Agilent Bioanalyzer instrument, using the Eukaryote Total RNA Nano kit to evaluate RIN and concentration. The sample libraries were prepared as described in "Procedure & Checklist – Iso-Seq™ Express Template Preparation for Sequel® and Sequel II Systems" (PN 101-763-800 Version 02 (October 2019)) using the NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module, the Iso-Seq Express Oligo Kit, ProNex beads (Promega) and the SMRTbell Express Template Prep Kit 2.0. 300 ng of total RNA was used for cDNA Synthesis followed by 12 + 3 cycles of cDNA Amplification. In the purification step of amplified cDNA the standard workflow was applied (sample is composed primarily of transcripts centered around 2 kb). Single cell/nuclei: Sequencing libraries were generated with unique dual indices (TT set A) and pooled for sequencing on a Novaseq6000 using a 100-cycle kit and 28-10-10-90 reads. Bulk RNAseq (truseq): Libraries were generated using Illumina TruSeq Stranded mRNA library prep kit (poly-A selection) and sequenced on a NextSeq500 (PE 2×150bp). Bulk RNAseq (pacbio): After purification the amplified cDNA went into the SMRTbell library construction. Quality control of the SMRTbell libraries was performed with the Qubit dsDNA HS kit and the Agilent Bioanalyzer High Sensitivity kit. Primer annealing and polymerase binding was performed using the Sequel II binding kit 2.0. Finally, the samples were sequenced on Sequel II and Sequel IIe System using Sequel® II Sequencing Plate 2.0, with an On-Plate Loading Concentration of 110 pM, movie time 24 hours and pre-extension time 2 hours.