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| Status |
Public on Mar 03, 2023 |
| Title |
Control 501, frontal lobe, bulk RNAseq |
| Sample type |
SRA |
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| Source name |
brain
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| Organism |
Homo sapiens |
| Characteristics |
tissue: brain
condition: control
age: 87
Sex: M
time post_injury: NA
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| Extracted molecule |
polyA RNA |
| Extraction 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).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Illumina Truseq
ERV_count_matrix_2.csv
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| Data processing |
Single nuclei RNAseq: Raw base calls were demultiplexed to obtained sample specific FastQ files and reads were aligned to GRCh38 genome assembly using the Cell Ranger pipeline (10x Genomics, Cellranger count v5) with default parameters (--include-introns was used for nuclei mapping)
Single nuclei RNAseq: Downstream analyses was performed with Seurat. All samples were merged and batch effects were removed using the Harmony algorithm. Cells with at least 1000 detected genes were retained and the data was normalized to transcript copies per 10,000, and log-normalized to reduce sequencing depth variability.
Single nuclei RNAseq: For visualization and clustering, manifolds were calculated using UMAP methods (RunUMAP, Seurat) and 20 precomputed principal components and the shared nearest neighbor algorithm modularity optimization-based clustering algorithm (FindClusters, Seurat) with a resolution of 0.1.
Single nuclei RNAseq: trusTEr (https://github.com/raquelgarza/truster) was ran pooling cells within clusters per condition, and per sample per condition.
Bulk RNAseq: Unique mapping using STAR aligner (--outFilterMultimapNmax 1, --outFilterMismatchNoverLmax 0.03)
Bulk RNAseq: ERV predictions from RetroTector (https://github.com/PatricJernLab/RetroTector) were quantified using featureCounts (Subread version 1.6.3), forcing matching strandness of the reads to the features being quantified (-s 2).
Bulk RNAseq: BAM files were indexed using samtools (version 1.12) and converted to bigwig files using deeptools bamCompare (version 2.5.4) (--normalizeUsingRPKM)
Assembly: GRCh38
Supplementary files format and content: Single nuclei RNAseq: Cellranger's filtered_feature_bc_matrix: barcodes, features and matrix files
Supplementary files format and content: Single nuclei RNAseq: csv files containing TE subfamily quantification per cluster per condition (tbi and control _TE_raw_matrix.csv). This is basically Tetranscripts output reformatted into csv
Supplementary files format and content: Single nuclei RNAseq: csv files containing TE subfamily quantification per cluster per sample ([sample] _TE_raw_matrix.csv). This is basically Tetranscripts output reformatted into csv
Supplementary files format and content: Single nuclei RNAseq: csv files containing TE subfamily quantification per cell in cluster (cluster expression / num cells in cluster), per condition (tbi and control _TE_norm_cluster_size_matrix.csv)
Supplementary files format and content: Single nuclei RNAseq: csv files containing TE subfamily quantification per cell in cluster (cluster expression / num cells in cluster), per sample ([sample] _TE_norm_cluster_size_matrix.csv)
Supplementary files format and content: Bulk RNAseq bigwigs for genome browser visualization
Supplementary files format and content: Bulk RNAseq ERV quantification read count matrix
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| Submission date |
Jul 22, 2022 |
| Last update date |
Mar 03, 2023 |
| Contact name |
Johan Jakobsson |
| E-mail(s) |
johan.jakobsson@med.lu.se
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| Organization name |
Lund University
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| Department |
Wallenberg Neuroscience Center
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| Lab |
Molecular Neurogenetics
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| Street address |
Sölvegatan 17
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| City |
Lund |
| ZIP/Postal code |
22362 |
| Country |
Sweden |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE209552 |
Single-cell transcriptomics of resected human traumatic brain injury tissues reveals acute activation of endogenous retroviruses in oligodendroglia |
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| Relations |
| BioSample |
SAMN29900436 |
| SRA |
SRX16459251 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6376808_DA656_TBI_ctrl_501F_S16_Aligned.sortedByCoord.out.bw |
80.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
| Raw data are available in SRA |
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