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Status |
Public on Jan 16, 2023 |
Title |
um_berninger_2019_01_003_p1_C01_1c_S3 |
Sample type |
SRA |
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Source name |
adult neural stem cell
|
Organism |
Mus musculus |
Characteristics |
cell type: adult neural stem cell treatment: control virus days post-treatment: 3
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Growth protocol |
Mice were administrated with lentivirus which encode EGFP or Yap1 5SA. DG was dissected from hippocampus and the tissue was enzymatically dissociated in Neural Tissue Dissociation Kit (Miltenyi Biotec, Germany). Dissociation was stopped by adding 10 ml HBSS (Ca+, Mg+) (Invitrogen). Cells were filtered by 70 µm cell strainer, then centrifuged at 300 g for 10 min. Single cell suspension was resuspended with PBS which contains 5% FBS.
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Extracted molecule |
polyA RNA |
Extraction protocol |
All the EGFP positive cells were collected into lysis buffer of 96-wells plates directly by Aria II SORP cell sorter at single cell sorting mode. Libraries were prepared using the Illumina Nextera XT DNA sample preparation kit. The protocol is based on the Tagmentation protocol from Fluidigm, which uses ¼ of the reagents per tagmentation reaction with a starting amount of 100-125pg of cDNA. Through this tagmentation step, the cDNA is fragmented and partial Nextera adaptors are added to each end of the cDNA fragments. A PCR follows, which adds the rest of the Nextera adaptors and the i5 and i7 dual indexes. Libraries were amplified in 12 PCR cycles. Three microliters of each library were pooled into a single tube and the pool was subsequently bead purified using Agencout AMPure XP beads, with a ratio of 0.9:1 Beads:DNA. The purified pool was profiled in a High Sensitivity DNA chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). Four pools (coming from four 96-well plates) are again pooled together in equimolar ratio and sequenced in a single NextSeq 500 Highoutput Flowcell, single read for 1x 75 cycles for read 1 plus 2x 8 cycles for each index read. scRNA-Seq
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Description |
Control-d03 expression of control virus in adult neural stem cells for 3 days
|
Data processing |
Smart-Seq2 raw data demultiplexing was performed using Illumina’s bcl2fastq conversion software v.2.19.1 and overall sequence quality was assessed with FastQC v0.11.5. . STAR v.2.5.2b with default parameters (except –outFilterMismatchNoverLmax 0.04) was used to align reads to the mouse reference genome GRCm38 (mm10), which was supplemented with ERCC RNA Spike-In Mix (ThermoFischer) control sequences and the GTF gene annotation from Ensembl release 88. Read summarization at the gene level was performed using Subread featureCounts v.1.6.2 with default parameters. Genome_build: GRCm38 with ERCC control sequences Supplementary_files_format_and_content: Raw count matrix with genes in rows and cells in columns
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Submission date |
Feb 07, 2020 |
Last update date |
Jan 16, 2023 |
Contact name |
Gregorio Alanis-Lobato |
E-mail(s) |
g.alanis.lobato@gmail.com
|
Organization name |
Boehringer Ingelheim Pharma
|
Department |
Global Computational Biology and Digital Sciences
|
Street address |
Birkendorfer Str 65
|
City |
Biberach an der Riss |
ZIP/Postal code |
88400 |
Country |
Germany |
|
|
Platform ID |
GPL13112 |
Series (1) |
GSE144967 |
The transcriptional co-activator Yap1 promotes adult hippocampal neural stem cell activation |
|
Relations |
BioSample |
SAMN14066362 |
SRA |
SRX7693383 |