|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on May 10, 2024 |
Title |
Spinal cord, Uninjured, P6981_1009 |
Sample type |
SRA |
|
|
Source name |
Spinal cord
|
Organism |
Mus musculus |
Characteristics |
tissue: Spinal cord cell type: Perivascular cells genotype: GLAST-CreERT2;R26R-tdTomato;Pdgfrb-eGFP Sex: female sorted population: EGFP+; tdTomato-
|
Extracted molecule |
total RNA |
Extraction protocol |
Preparation of single-cell cell suspension and myelin removal was adapted from previously published protocols (Lovatt et al., 2007; Orre et al., 2014). In summary, the procedure was: 5 days after spinal cord crush injury, animals were euthanized by intraperitoneal injection of sodium pentobarbital (200mg/Kg, 100ul i.p., APL) and transcardially perfused with cold Hank's Balanced Salt Solution without calcium or magnesium (HBSS) (Invitrogen). The vertebral column was immediately removed to cold HBSS, the spinal cord was carefully dissected out of the vertebrae and the crush injury segment was separated from the uninjured spinal cord and treated separately hereafter. After removal of the outer meningeal layers, the tissue was dissociated mechanically with a razor blade on a glass petri dish and immediately transferred to cold HBSS. Followed by enzymatic digestion with papain at a final concentration of 8 U/mL with 80 Kunitz units/mL DNase I (Sigma-Aldrich, D4263) in Ca2+/Mg2+-free piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES)/cysteine -based buffer, pH 7.4, for 40min at 37°C, 350rpm on a ThermoMixer® (Eppendorf). The tissue was carefully triturated and incubated for additional 5min. The cell suspension was passed through a 70μmCell Strainer (Corning), washed with Minimum Essential Media (MEM) with 1% bovine serum albumin (BSA) and spun down at 200g for 5min at 4°C. The cells were resuspended in MEM and centrifuged over a 90% Percoll gradient (GE Healthcare, 17-0891-01) at 250g for 15min at 4°C. Cells in the lipid layer and below were diluted 5 times in MEM with 1% BSA and spun in a 15mL tube at 250g for 10min at 4°C. All supernatant including the lipid layer were carefully removed and the pellet resuspended in cold MACS Buffer and magnetic Myelin removal beads (Miltenyi biotech, 130-096-433) and incubated for 15min at 4°C. The cells were washed and run over MACS MS columns (Miltenyi biotech, 130-042-201) on a magnetic stand according to the manufacturer’s instructions. The flow through cells were collected, spun down and resuspended in FACS buffer. The single cell suspensions derived from normal spinal cord and injury segments were subject to FACS (fluorescence activated cell sorting) into GLAST-CreER-tomato+/Pdgfrb-eGFP+ double positive and Pdgfrb-eGFP+ single cells, into 96-well plates with Cell lysis buffer (Clonetech, 635013) and frozen to -80°C. Cell sorting was performed on a FACSAria III Cell Sorter system (BD Biosciences). Singlet discrimination was performed using plots for forward scatter (FSC-A versus FSC-H) and side scatter (SSC-W versus SSC-H), dead cells were excluded by SYTOX Blue Dead Cell Stain (ThermoFisher Scientific, S34857). Single cells were processed to cDNA libraries according to the Smart-Seq2 protocol (Picelli et al. 2014). Tn5 tagmentation was performed using the Nextera XT DNA library Preparation Kit (Illumina, FC-131-1002) using dual indexes (i7 and i5).
|
|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
|
|
Data processing |
Clustering was done by 'cBot' and samples were sequenced on Illumina HiSeq2500 (HiSeq Control Software 2.2.58/RTA 1.18.64) with a 1x51 setup using 'HiSeq SBS Kit v4' chemistry. The Bcl to FastQ conversion was performed using bcl2fastq from the CASAVA software suite. The quality scale used is Sanger / phred33 / Illumina 1.8+. Mus musculus, GRCm38 was used as reference genome. We used a single-cell RNA sequencing snakemake workflow (“lts_workflows_sm_scrnaseq”) for alignment, quantification and quality control using resources as follows: The reads were aligned to the mouse (mm10) genome using STAR 2.5.3a (Dobin et al., 2013) and transcripts quantified in terms of Reads Per Kilobase of transcript per Million mapped reads (RPKM) using rpkmforgenes v1.0.1 (Ramsköld et al., 2009) and RSEM to estimate gene and isoform expression levels v1.2.28 (Li and Dewey, 2011). RSeQC V2.6.4 (Wang et al., 2012) Multiqc v0.9.1 were used for quality control. To filter out low quality libraries the following filtering criteria for quality control were applied: star_Uniquely mapped: < mean - 2 sd, gene_detection : > mean - 2 sd & < mean + 2 sd, count_gene : < mean - 2 sd, gene_correlation_max : < mean - 2 sd. Cells that failed for 2 of the criteria were filtered out (25 cells, 2.89% of 864, the total number of cells). Assembly: mm10 Supplementary files format and content: rpkmforgenes_counts.tab Supplementary files format and content: 170706_sampleinfo_scs.csv
|
|
|
Submission date |
Apr 17, 2023 |
Last update date |
May 10, 2024 |
Contact name |
Christian Göritz |
E-mail(s) |
christian.goeritz@ki.se
|
Organization name |
Karolinska Institutet
|
Department |
CMB
|
Street address |
Solnavägen 9
|
City |
Solna |
ZIP/Postal code |
17165 |
Country |
Sweden |
|
|
Platform ID |
GPL17021 |
Series (1) |
GSE229916 |
Pericytes and perivascular fibroblasts contribute to central nervous system fibrosis in a region dependent manner |
|
Relations |
BioSample |
SAMN34225586 |
SRA |
SRX19993360 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
|
|
|
|
|