|
| Status |
Public on Jun 04, 2018 |
| Title |
AW6-CD1-BC2.2 |
| Sample type |
SRA |
| |
|
| Source name |
mammary gland
|
| Organism |
Mus musculus |
| Characteristics |
tissue: mammary gland
gender: female
mouse strain: CD1
cell surface markers: CD45-CD31-CD140a-CD24+CD29hign
|
| Treatment protocol |
Targeting Confetti, tdTomato or ΔNp63-IRES-GFP expression in the MG – For clonal lineage tracing, K14rtTA/TetO-Cre/Rosa-Confetti embryos were induced at E13 by intravenous (IV) injection in the tail vein of the pregnant mother with 1µg/g of Doxycycline (diluted in sterile PBS, Sigma) and sacrificed 2 days later (E15) or 5 days after delivery (P5). K5CreER/Rosa-Confetti newborn pups (P1) were induced by intraperitoneal (IP) injection of 50µg of Tamoxifen (diluted in sunflower seed oil, Sigma) and sacrificed 21 days later. For lineage tracing at saturation, K14rtTA/TetO-Cre/Rosa-tdTomato or K14rtTA/TetO-Cre/Rosa-ΔNp63-IRES-GFP embryos were induced at E13 by IV injection in the tail vein of the pregnant mother with 15µg/g of Doxycycline (diluted in sterile PBS) and sacrificed 5 days after delivery (P5). K8rtTA/TetO-Cre/Rosa-Confetti female mice were induced at 4w old with Doxycyclin treatment during 7 days consisting in the combination of Doxycylin 10g/kg in diet (Bio-Serv), 2g/l in drinking water (AG Scientific) and 3 intraperitoneal injections of 2 mg in 200µl PBS.
|
| Growth protocol |
Mice – Lgr5-EGFP-IRES-CreER18 and Rosa-tdTomato63 mice were obtained from the Jackson Laboratory. Rosa-Confetti15 mice were provided by H. Clevers; K14rtTA transgenic mice64 were provided by Elaine Fuchs; TetO-Cre mice65 were provided by Andreas Nagy; Rosa26-ΔNp63-IRES-GFP mice57 were provided by Wim Declercq. The generation of K5CreER and of K8rtTA were previously described9, 66. All experimental mice used in this study were females of mixed genetic backgrounds. No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Mice colonies were maintained in a certified animal facility in accordance with European guidelines. The experiments were approved by the local ethical committee (CEBEA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNAseq and analysis of bulk samples40000 LCs and 5000 BCs were isolated by FACS as described above and collected into kit lysis buffer. RNA was extracted using absolutely RNA nanoprep kit (Stratagene).
RNA quality was checked using a Bioanalyzer 2100 (Agilent technologies). Indexed cDNA libraries were obtained using the Ovation Solo RNA-Seq System (NuGen) following manufacturer recommendation. The multiplexed libraries (18 pM) were loaded on flow cells and sequences were produced using a NovaSeq 6000 S2 Reagent Kit (200 cycles) from a NovaSeq 6000 System (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
CP20M-Wuidart
|
| Data processing |
Approximately 19 million of paired-end reads per sample were mapped against the mouse reference genome (GRCm38/mm10) using STAR software to generate read alignments for each sample. Annotations Mus_musculus.GRCm38.87.gtf were obtained from ftp.Ensembl.org. After transcripts assembling, gene level counts were obtained using HTSeq. Fold change of mean gene expression for the duplicates were used to calculate the level of differential gene expression. Heatmap of the 500 most variable genes across the 8 samples and corresponding clustering dendrogram were drawn with heatmap.2 function of the R package gplots (citation R). Euclidian distance with complete linkage agglomeration method was used for clustering.
Genome_build: grcm38
Supplementary_files_format_and_content: CP20M-Wuidart (count normalized by 20M mapped reads
|
| |
|
| Submission date |
Jan 26, 2018 |
| Last update date |
Jun 04, 2018 |
| Contact name |
Alexandra Van Keymeulen |
| Organization name |
Université Libre de Bruxelles
|
| Department |
IRIBHM
|
| Lab |
Blanpain
|
| Street address |
808, route de Lennik CP602
|
| City |
Bruxelles |
| ZIP/Postal code |
1070 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE109707 |
Early lineage segregation of multipotent embryonic mammary gland progenitors [RNA-seq I] |
| GSE109711 |
Early lineage segregation of multipotent embryonic mammary gland progenitors |
|
| Relations |
| BioSample |
SAMN08400241 |
| SRA |
SRX3602394 |
