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| Status |
Public on Dec 14, 2020 |
| Title |
TLSMG 120h rep2 |
| Sample type |
SRA |
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| Source name |
TLSMG (6x pooled)
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| Organism |
Mus musculus |
| Characteristics |
cell type: mESC
strain: F1G4
genotype: T::H2B-mCherry/Sox2::H2B-Venus double reporter
tissue: 6 pooled TLSMG (gastruloids at 120h in matrigel)
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| Growth protocol |
mESCs culture: Mouse embryonic stem cells (mESCs) were cultured on 6 cm plates gelatinized with 0.1% gelatin and coated with feeder cells (3-4x10^4cells/cm²) in ES+LIF medium at 37 °C and 5% or 7.5% CO2. mESCs were split every second day with a dilution suitable to the proliferation velocity (between 1/5 and 1/9). ES+LIF medium was refreshed daily. For splitting, media was aspirated and cells were washed once with PBS and trypsinized for 5-10’ at 37 °C. Trypsin was neutralized by 3 ml ES+LIF and centrifuged for 5’ at 1000 rpm, after which the pellet was resuspended in ES+LIF. For freezing of mESCs, cell pellets were resuspended in ES medium with 20% FCS, and mixed in a 1:1 ratio with ES freezing medium. Cells were frozen down o/n in the –80 °C and transferred to liquid nitrogen the next day.
Generation of gastruloids: Gastruloids were generated as described previously (Beccari et al., Nature 2018), with some minor modifications. First, mESCs were feeder freed. To this end, mESCs were trypsinized on the feeder plate as described above, washed with ES+LIF and resuspended in 2ml ES+LIF. On three gelatinized (0.1% gelatin) wells of a 6-well plate, cells were sequentially plated for 25’, 20’ and 15’. With each transfer, cells were triturated to maintain a single cell suspension. Feeder-freed mESCs were then washed once in 5 ml PBS containing MgCl2 and NaCl and once in 5 ml NDiff227. mESCs were then pelleted by centrifugation for 5’ at 1000rpm and resuspended in 500 µl of N2B27. 10 µl of the cell suspension was mixed with 10 µl of Trypanblue (Bio-Rad) for automated cell counting with Luna Automated Cell Counter. 200-250 live cells were then plated in a volume of 30 to 40 µl NDiff227 into each well of a 96-well round bottom, low attachment plate (Cellstar 96 well suspension culture plate or Costar 7007 ultra-low attachment 96 well plate). Cells were then allowed to aggregate for 48 h. After these 48h cells were pulsed with 3 µM CHIR99021 (CHI) in 150 µl of NDiff227. Between 72 and 120h aa, medium was refreshed every 24 h by removing 150 µl of the old media and adding the same volume of new, pre-incubated NDiff227. For the "96h" samples cells harvested at 96h aa and for the "Gastruloid" at 120h aa and subjected to bulk RNA sequencing and for 96h also for single cell RNAseq ("TLS precursor").
Generation of TLS: The gastruloid protocol described above was followed until 96h aa. Gastruloids were then embedded in 5% Growth-Factor-Reduced Matrigel (MG) (Corning). To this end, fresh NDiff227 medium was pre-incubated for at least 20’ at at 37 °C and 5% or 7.5% CO2. Pre-incubated medium was then put on ice for 5’, after which MG was added to achieve a final concentration of 5%. Medium was then put on room temperature for 5’, during which 150µl of old medium was removed from the aggregates. New medium with 5%MG (150µl) was then added, and the cultures were returned to the incubator and further cultured at 37 °C and 5% or 7.5%. Cultured were allowed to settle for at least 30’ before proceeding to further experimentation (e.g. live imaging). For TLS treated with CHI ("TLSMGC") and CHI+LDN ("TLSMGCL"), 5 µM CHI with or without 600 nM LDN was added from 96h to 120h aa prior to adding the MG. For controls ("TLSMG"), an equal volume of diluent (DMSO) was added. All conditions were subjected to bulk RNAseq and the TLSMG were subjected to single cell RNAseq at 108h and 120h aa.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Structures have been generated as described below. The experiment has been performed in 3 biological replicates . 6 structures per replicate have been selected (based on the presence of an asymmetric pole) and pooled in a tube. All samples have been washed twice with 1xPBS/0.4%BSA. Then 350ul of RLT Plus buffer containing 1% β-mercaptoethanol (Thermo Cat. No. 21985023) have been added to dissociate the structures and lysate the cells. After pipette dissociation and vortexing, samples have been frozen at -80C. The following day, RNA has been extracted using RNeasy Plus Micro Kit (Qiagen Cat No./ID: 74034) and RNA concentration and quality has been established using Agilent RNA 6000 Pico kit (5067-1513) on an Agilent 2100 Bioanalyzer. All samples analyzed had a RINe values above 8.0, and were then used for library prep.
mRNA libraries have been prepared using KAPA Stranded RNA-Seq Kit (KapaBiosystem 07962142001) following the standard manual protocol. 500ngs of total RNA have been used for each sample to enter the library preparation protocol. For adapter ligation dual indexes have been used (NEXTFLEX® Unique Dual Index Barcodes NOVA-514150) at a working concentration of 71nM (5ul of 1uM stock in each 70ul ligation reaction). Quality and concentration of the obtained libraries have been measured using Agilent High Sensitivity D5000 ScreenTape (5067-5588) on an Agilent 4150 TapeStation. All libraries have been sequenced using 75bp-paired end sequencing (150 cycles kit; FC-410-1002) in a HiSeq4000 platform at a minimum of 23,7 million fragments per sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Bulk RNA-sequencing of 6 pooled TLSMG (gastruloids at 120h in matrigel)
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| Data processing |
RNAseq reads were pre-processed using cutadapt v2.4 to remove adapter and trim low quality bases.
Reads were subsequently aligned against the reference gnenome mm10 using STAR v2.7.3a (parameter: outSAMtype BAM SortedByCoordinate --outSAMattributes Standard --outSAMstrandField intronMotif --outSAMunmapped Within --quantMode GeneCounts).
Stringtie v2.0 was used for transcript quantification, e.g. calculation of strand-specific coverage, RPKMs and TPMs (reference: mm10 gencode gene annotation gtf).
genome build: mm10
Tab-delimited file containing gene abundances: <Gene ID> <Gene Name> <Reference> <Strand> <Start> <End> <Coverage> <RPKM> <TPM>
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| Submission date |
Nov 29, 2019 |
| Last update date |
Dec 14, 2020 |
| Contact name |
Helene Kretzmer |
| E-mail(s) |
[email protected]
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| Organization name |
Max Planck Institute for Molecular Genetics
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| Department |
Genome Regulation
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| Lab |
Meissner Lab
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| Street address |
Ihnestraße 63
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| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE141175 |
Self-organized trunk-like-structures with somites and a neural tube |
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| Relations |
| BioSample |
SAMN13428596 |
| SRA |
SRX7241731 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4196557_TLSMG_rep2.stringtie.tsv.gz |
1.3 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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