 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 05, 2022 |
| Title |
EBs.MUA_EV_004_X374 |
| Sample type |
SRA |
| |
|
| Source name |
embryoid bodies from mouse ESCs
|
| Organism |
Mus musculus |
| Characteristics |
celseq2 adapter: BC_374
sample type: XEN
|
| Growth protocol |
Advanced N2B27 medium was used as the basic medium consisting of 2% B27 (Gibco, 17504-044) and 1% N2 (Gibco, 17502-048), a 1/1 mixture of Advanced DMEM/F12 (ThermoFisher, 12634028) with Neurobasal (ThermoFisher, 12348017), 2 mM Glutamax (Gibco), 10mM NEAA (ThermoFisher, 11140050), 0.5% Bovine Serum Albumin (Sigma-Aldrich, A7979), 10 mM HEPES (Gibco, 15630056), 1 mM Sodium Pyruvate (ThermoFisher, 11360070). supplemented right before use with 10 ng/mL leukaemia inhibitory factor (Lif, Merck Millipore ESG1106), 1 ?M PD0325901 (AxonMed, 1408), 3 ?M CHIR99021 (AxonMed, 1386) and 50 ?M 2-mercaptoethanol (Gibco, 11528926). For ESC expansion the cells were expanded for minimally 2 passages on gelatin-coated tissue-culture plastic in advanced N2B27 medium was supplemented right before use with 3 ?M CHIR99021 (AxonMed 1386), 10 ng/mL Lif (Merck Millipore ESG1106), 1 ?M PD0325901 (AxonMed 1408) and 50 µM 2-mercaptoethanol (Gibco 11528926).
PrE-induction for Rosette formation was done by seeding an average of 14 ESC per microwell in Advanced B27N2 medium supplemented with 10 mM HEPES and penicillin/streptomycin, and the PrE-induction compounds 3 ?M CHIR99021 (AxonMed 1386), 50 ng/mL Fgf4 (RnD systems, 5846-F4-025), 10 nM RA (Sigma R2625), 1 mM 8Br-cAMP (Biolog, B007-50E), 1 µg/mL heparin (Sigma) and 50 µM 2-mercaptoethanol (Gibco 11528926). When seeding the cells in the microwells 2 µM Y27632 (AxonMed 1683) was added. Microwell arrays were pre-wetted in serum-medium containing 100U/ml penicillin/streptomycin before use. After 24 h or 48 h of culture, cells were washed once with advanced N2B27 medium and then refreshed with advanced N2B27 supplemented with 50 µM 2-mercaptoethanol. After 72 h EBs were flushed out and transferred into non-TCPS 6-well plates with 2 mL advanced N2B27 medium supplemented with 50 µM 2-mercaptoethanol.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
EBs were washed twice in PBS before collagenase IV (600 U/ml) was added. Plates were shaken at 350 RPM for 30 minutes before being transferred to TrypLE 10X (Thermofisher, A1217701, no dilution) and shaken again for 20 minutes. EBs were dissociated into single cell suspensions using a small capillary. The resulting suspension was quenched using PBS with 50% FBS, centrifuged and resuspended in 230 µl of PBS with 10% FBS. Cells were stained with Pdgfr? antibody (1:150 dilution) for 30 min at 4°C followed by three PBS (+10%FBS) washes, secondary antibody incubation (1:400) for 30 min. Cells were washed three times in PBS and sorted for further processing.
Rosettes were manually picked and placed in a round-bottom 96wp-well containing 100 µl PBS + 0.5% BSA. When sufficient numbers were collected, they were transferred to an Eppendorf tube with 600 µl Accumax and incubated at 37 degree C for 30 min. Every 5-10 minutes the mixture was resuspended using a 200 µl pipette. When single cells were observed under the microscope the suspension was centrifuged at 200g for 4 min and, supernatant was removed and cells were resuspended in staining solution in PBS (2 µM Di-I + 0.1% Dead-stain-647 ) and incubated for 20 min at RT in the dark. Epiblast-only rosettes were washed once with PBS before domes were disrupted using 150 µl of a 1:1 DipaseII:N2B27 mixture and incubated for 20 min at 37 degree C in a 1.5ml Eppendorf tube (4 domes per tube). Solution was resuspended every 5-10 minutes using a 200 µl pipette tip. Finally, the cell suspension was resuspended in 700 µl Accumax with 100 µl of PBS with 0.5% BSA and incubated for 25 min at 37 degree C. Single cells were stained with 2 µM Di-I + 0.1% Dead-stain-647) and incubated for 20 min at RT in the dark.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Transcriptome measurement of sample EBs.MUA_EV_004_X374
|
| Data processing |
Demultiplexing of raw data (previously demultiplexed on Illumina index) was done using barcodes. Read1 measurements contained only the barcode and UMI followed by unalignable sequence , usually polyT and are therefore not included. Read2 of the transcriptomic measurements contains the mRNA sequence. All reads provided are read 2. The sequencing was run in 4 lanes: L001-L004.
mRNA reads (in read2) were aligned using tophat2 (v2.1.1) using parameters ‘--segment-length 22 --read-mismatches 4 --read-edit-dist 4 --min-anchor 6 --min-intron-length 25 --max-intron-length 25000 --no-novel-juncs --no-novel-indels --no-coverage-search --b2-very-sensitive --b2-N 1 --b2-gbar 200’ to a transcriptome (options ‘--GTF’ and ‘--transcriptome-index’) obtained from GENCODE (v26) supplemented with ERCC mRNA spike-in sequences.
Reads and unique UMIs were counted per gene (according to the “intersection-strict” algorithm of `htseq-count` [http://htseq.readthedocs.io/en/master/count.html]). For each gene, the number of unique observed UMIs was trimmed by starting at the UMI with highest read count and setting UMIs with Hamming distance of 1 to zero, and iteratively filtering all UMI sequences (in case of equal read counts for a UMI, ties were broking by lexicographic sorting of the UMIs). The number of observed distinct UMIs after trimming was taken as the transcript count for that gene.
Genome_build: mm10
Supplementary_files_format_and_content: Tables are provided with the number of transcripts per gene.
|
| |
|
| Submission date |
Mar 04, 2022 |
| Last update date |
Mar 07, 2022 |
| Contact name |
Isabel Guerreiro |
| E-mail(s) |
[email protected]
|
| Organization name |
Hubrecht Institute
|
| Street address |
Uppsalalaan 8
|
| City |
Utrecht |
| ZIP/Postal code |
3584CT |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE129655 |
The chemically-defined induction of a primitive endoderm and epiblast-like niche supports post-implantation progression from blastoids. |
|
| Relations |
| BioSample |
SAMN26437335 |
| SRA |
SRX14370212 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5933911_EBs.MUA_EV_004_X374.tsv.gz |
160.6 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
