 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 13, 2017 |
| Title |
Cwt_pc5_+_HJag |
| Sample type |
SRA |
| |
|
| Source name |
Cell lines
|
| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
cell line (receptor side): C2C12 cells
mouse cell genotype/variation: wild type
cocultured with: human HEK-293-Flp-In cells
ligand side: Jag1 WT (Flp Jag1+)
human cell genotype/variation: Jag1 expression
|
| Treatment protocol |
In one 12-well plate, we seeded 3 wells of mouse C2C12 control cells and 3 wells of C2C12-FLN1 cells, with 3.6x105 cells in 1 mL antibiotic-free medium per well. Cells were allowed to settle for 8 hours. C2C12 control and C2C12-FLN1 cells were transfected with pcDNA5 (1.6 ug/well). All transfections were done using Lipofectamine® 2000 (InvitrogenTM, cat. no. 11668-019) with Opti-MEM® I Reduced Serum Medium (Gibco®, cat. no. 31985-062), according to manufacturer’s instructions. The following day (18 hours post transfection), 3.6x105 cells in 0.5 mL antibiotic-free medium of Flp Ctrl, Flp Jag1+, or Flp Jag1Ndr cells were added. Cells were co-cultured for 6 hours, then lysed in 350 L per well Buffer RLT (QIAGEN, cat. no. 79216) with 1% 2-Mercaptoethanol (Sigma-Aldrich®, cat. no. M3148) and stored at -80°C until RNA extraction.
|
| Growth protocol |
Mouse C2C12 control and C2C12-FLNotch1 (latter with 1ug/mL puromycin selection; Puromycin dihydrochloride from Streptomyces alboniger, Sigma-Aldrich®, cat. no. P7255), and human HEK-293-Flp-In cells (Hansson et al., 2010): HEK293-Flp control (Flp Ctrl), HEK293-Flp-Jag1WT (Flp Jag1+), HEK293-Flp-Jag1Ndr (Flp Jag1Ndr) (latter two with 300 ug/mL hygromycin selection; Hygromycin B, Gibco®, cat. no. 10687-010) were cultured in complete medium, composed of DMEM, high glucose, pyruvate (Gibco®, cat. no. 41966-029) supplemented with 10% fetal bovine serum (Gibco®, cat. no. 10270-106) and 1% penicillin-streptomycin (10,000 U/mL, Gibco®, cat. no. 15140-122), at 37°C in a humidified 5% CO2 atmosphere.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA extraction for all cell lysates from the two-species co-culture and immobilized ligand assay was performed using the RNeasy Mini Kit (cat. no. 74104, QIAGEN), including on-column DNase I digestion (cat. no. 79254, QIAGEN).
cDNA libraries for all samples were created using the TruSeq® RNA Sample Prep Kit v2–48, Set A (cat. no. RS-122-2001, Illumina®,) as per the TruSeq® RNA Sample Preparation v2 Guide, Low-Throughput Protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
RPKM Cwt_pc5_+_HJag
|
| Data processing |
Reads were aligned using STAR to genomes mm9 and hg19
Reads were separated for different species using S^3 (https://github.com/danielramskold/S3_species-specific_sequencing) with settings -Ms 3 -Mu 3 -D 2
Reads that mapped uniquely in the target species' genome were selected
Gene expression values were calculated using rpkmforgenes.py (see link above) with options -rmnameoverlap -bothendsceil -u and using RefSeq annotation from 7 April 2013 and unique alignment positions from http://sandberg.cmb.ki.se/multo/
Genome_build: mm9 and hg19
Supplementary_files_format_and_content: Tab-delimited text of normalised expression values and read counts
|
| |
|
| Submission date |
Oct 12, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Urban Lendahl |
| Organization name |
Karolinska Insttitutet
|
| Street address |
Von Eulers vag 3
|
| City |
Stockholm |
| ZIP/Postal code |
17177 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL16512 |
| Series (2) |
| GSE104874 |
RNA Seq of C2C12 cells stimulated with Control, Jag1-expressing or Jag1Ndr-expressing cells |
| GSE104876 |
Alagille_Nodder |
|
| Relations |
| BioSample |
SAMN07776810 |
| SRA |
SRX3275373 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
