 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 06, 2021 |
| Title |
Astrocytes_IM2-GC F01 |
| Sample type |
SRA |
| |
|
| Source name |
Astrocytes
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: iPSC-derived astrocytes
genotype: Wild-type
platemap well: F01
cdna barcode: GTAGTTCG
|
| Treatment protocol |
The cells were not treated.
|
| Growth protocol |
Patient-derived iPSCs with the LRRK2 mutation G2019S and the corresponding isogenic control were provided by Prof. Dr. Thomas Gasser (Universitätsklinikum Tübingen) and Prof. Dr. Hans R. Schöler (Max-Planck Institute). The cells were genotyped to confirm the presence of the LRRK2 mutation G2019S using a TaqMan SNP genotyping assay (Thermo Fisher Scientific, Waltham, MA), and karyotyped every 2 months to confirm genomic stability (Cell Line Genetics, Madison, WI). The iPSCs were cultured in mTesR1 or mTesR Plus media (StemCell Technologies, Vancouver, Canada), regularly passaged as aggregates using ReLeSR (StemCell Technologies), and cultured on Geltrex-coated plates (Thermo Fisher Scientific). The step-by-step protocols, media recipes and quality control experiments to prepare iPSC-derived NPCs and astrocytes have been described in exhaustive details in de Rus Jacquet et al., Current Protocols in Cell Biology (2019), https://0-doi-org.brum.beds.ac.uk/10.1002/cpcb.98.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were homogenized in TRIzol and stored at -80 until processing
Total RNA was isolated according to the manufacturer’s instruction, quantified by Nanodrop (Thermo Fisher Scientific) and diluted to 1 ng/µl in nuclease-free water. A total of 1 ng of RNA was added to 2.5 µl of cell lysis buffer (nuclease-free water with 0.2 % v/v Triton X-100 and 0.1 U/µl RNase inhibitor), and subjected to cDNA synthesis and amplification as described before (Cembrowski et al., 2018). Libraries were prepared using a modified Nextera XT DNA protocol (Illumina, San Diego, CA) where 5 µM P5NEXTPT5 was substituted for the i5 primers in the kit. Libraries were quantified by qPCR (Roche), normalized to 2 nM, then pooled and sequenced on a NextSeq550 flowcell with 25 bases in read 1, 8 bases in the i7 index read, and 50 bases in read 2. The control library phiX (Illumina) was spiked in at a final concentration of 15% to improve color balance in read 1.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
Astrocytes_IM2-GC
Astrocytes_IM2-GC
|
| Data processing |
Smrtscrb2 analysis pipeline: Custom python scripts were used to extract Barcode and UMI sequences from read 1. The correction of barcode error was achieved using starcode v1.1 (Zorita et al., 2015) with the following additional parameters: “-d 1 -q --print_clusters”. Read 2 sequences were renamed using the error-corrected barcode from starcode and UMI sequences from read 1, and were aligned to the Homo sapiens GRCh38.p12 genome assembly and annotation from Ensembl (ensembl.org) using STAR (Dobin et al., 2013) with the following additional parameters: “--alignIntronMax 200000 --outSAMattributes All --outSAMunmapped Within --outSAMtype BAM Unsorted”. The validity of an alignment was defined by the unique alignment to an exon feature on the correct strand. Gene-level counts were created using valid alignments with at least 50% of the read aligned to an exon feature, using a custom python script to collapse UMIs by gene. All custom scripts are available by request.
All custom scripts are available by request.
Differential gene expression analysis was performed using EBseq v3.8 in R (Leng et al., 2013), with condition 1 being LRRK2 G2019S and condition 2 being WT. EBseq calculated a median normalization of the sequencing counts using the median of ratios methods (Anders & Huber, 2010). First, the geometric mean of the sequencing counts was calculated for each gene and across all samples to create a pseudo-reference sample, and the ratio of the sequencing counts to the pseudo-reference counts was calculated for every gene. Then, for each sample, the median value of all the ratios was taken as the normalization factor. Finally, the median normalized sequencing counts were calculated by dividing the sample’s sequencing counts by the sample’s normalization factor. A false discovery rate of 0.05 and a fold change threshold of 1.4 or 0.7 were used to identify dysregulated genes in LRRK2 G2019S vs WT astrocytes. A k-means clustering algorithm (k=3) was used to group genes into low, moderate and highly expressed transcripts based on their log10(1+median normalized counts) value, using ComplexHeatmap v3.11 in R (Z. Gu et al., 2016). Gene ontology enrichment analysis was done using the Database for Annotation, Visualization, and Integrated Discovery (DAVID v6.8, https://david.ncifcrf.gov/) (Huang da, Sherman, & Lempicki, 2009a, 2009b). A list of all genes detected in all the samples was exported for use as the background gene set in DAVID.
Genome_build: Homo sapiens Ensembl GRCh38.95
Supplementary_files_format_and_content: single Microsoft Excel file that includes raw count values for all 58768 genes for all 20 samples and a list of 752 differentially expressed genes of which 698 were found in the DAVID v6.8 database
|
| |
|
| Submission date |
Jun 18, 2020 |
| Last update date |
Oct 06, 2021 |
| Contact name |
Aurelie de Rus Jacquet |
| E-mail(s) |
aurelie.jacquet@crchudequebec.ulaval.ca
|
| Phone |
5813974933
|
| Organization name |
Universite Laval
|
| Street address |
2705 Boulevard Laurier
|
| City |
Quebec |
| State/province |
QC |
| ZIP/Postal code |
G1V 4G2 |
| Country |
Canada |
| |
|
| Platform ID |
GPL21697 |
| Series (1) |
| GSE152768 |
The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease |
|
| Relations |
| BioSample |
SAMN15314830 |
| SRA |
SRX8574180 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
