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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 16, 2021 |
| Title |
vasaplate-HEK293T-mESC |
| Sample type |
SRA |
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| Source name |
vasaplate-HEK293T-mESC
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
protocol/barcodes: VASA-seq
read format: Read 1 starts with a 6-nucleotide long UFI, followed by an 8 nucleotide long cell-specific barcode
plate format: human cells sorted inwells 1,3,5, etc(i.e. A1, A3, A5, etc.) and mouse cells sorted in 2,4,6, etc (A2, A4, A6, etc)
sample type: mixed human HEK293T and mouse embryonic stem cells
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| Extracted molecule |
total RNA |
| Extraction protocol |
vasaplate: Single cells were sorted by FACS into 384-well plates containing RT-primers and oil. Reagents were dispensed with a robot and plates incubated in a thermocycles after each dispensing. Briefly; 1) Lysis/fragmentation mix, 2) Poly(A)-tailing/end repair mix, 3) Reverse transcription mix, 4) Second strand synthesis mix. After second strand synthesis, wells were pooled and material was amplified with IVT. Amplified material was depleted of rRNA, second adapter was ligated and aRNA converted to cDNA. Full Illumina handles were added by PCR, and samples were sequenced.
vasadrop: Single-cells were co-encapsulated with barcoded inDrop v3 beads with with a lysis and RNA fragmentation buffer. The droplets were then incubated and further injected with a RNA repair and poly(A) tailing mix using a pico-injector. After incubation, the droplets were the injected with a reverse transcription mix and incubated. The. Droplets were then de-emulsified and processed similarly to the VASA-plate workflow.
cell dissociation: HEK293T were cultured in DMEM, dissociated with Trypsin-EDTA and washed with ice-cold PBS. Mouse ES cells were cultured in 2i/LIF conditions, dissociated with Accutase and washed with ice-cold PBS. Mouse embryos from E8.5 and E9.5 were cut into pieces under the microscope before collection in a tube, embryos from stage E6.5 and E7.5 were not cut. TrypLE Express was used for cell dissociation and washed with PBS supplemented with 0.4% BSA.
Illumina TruSeq adapaters
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Sequencing libraries were mapped with STAR-2.5.3a with default parameters to the ensembl version 93 of the mouse genome, and only reads mapping to gene bodies (exons or introns) were used for downstream analysis. Reads mapping simultaneously to an exon and to an intron were assigned to the exon.
Genome_build: Ensembl version 99 for both mouse and human genomes
Supplementary_files_format_and_content: Tabular-separated file indicating number of UFIs per cell. In "total.UFICounts.tsv.gz" spliced and unspliced transcripts are reported together, and multi-annotations are indicated as "geneA_geneB". In "uniaggGenes_spliced.UFICounts.tsv.gz" and "uniaggGenes_unspliced.UFICounts.tsv.gz", count tables for spliced and unspliced transcripts are provided separatedy, and multi-annotations are aggregated.
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| Submission date |
Jun 10, 2021 |
| Last update date |
Sep 16, 2021 |
| Contact name |
Anna Alemany |
| E-mail(s) |
annalemany@gmail.com, a.alemany@lumc.nl
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| Phone |
+31638680750
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| Organization name |
LUMC
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| Department |
Anatomy and Embryology
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| Street address |
ALBINUSDREEF 2
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| City |
Leiden |
| State/province |
South Holland |
| ZIP/Postal code |
2333 ZA |
| Country |
Netherlands |
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| Platform ID |
GPL25526 |
| Series (1) |
| GSE176588 |
Droplet-based Single-cell Total RNA-seq Reveals Differential Non-Coding Expression and Splicing Patterns during Mouse Development |
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| Relations |
| BioSample |
SAMN19659009 |
| SRA |
SRX11116362 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5369495_bc2cellID_vasaplate.tsv.gz |
2.1 Kb |
(ftp)(http) |
TSV |
| GSM5369495_vasaplate_HEK293T-mESC_split_total.UFICounts.tsv.gz |
19.0 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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