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Status |
Public on Oct 13, 2017 |
Title |
CN_pc5_+_Hwt |
Sample type |
SRA |
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Source name |
Cell lines
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Organisms |
Homo sapiens; Mus musculus |
Characteristics |
cell line (receptor side): C2C12 cells mouse cell genotype/variation: Notch1 expression cocultured with: human HEK-293-Flp-In cells ligand side: control (Flp Ctrl) human cell genotype/variation: wild type
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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.
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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.
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Description |
RPKM CN_pc5_+_Hwt
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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
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Submission date |
Oct 12, 2017 |
Last update date |
May 15, 2019 |
Contact name |
Urban Lendahl |
Organization name |
Karolinska Insttitutet
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Street address |
Von Eulers vag 3
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City |
Stockholm |
ZIP/Postal code |
17177 |
Country |
Sweden |
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Platform ID |
GPL16512 |
Series (2) |
GSE104874 |
RNA Seq of C2C12 cells stimulated with Control, Jag1-expressing or Jag1Ndr-expressing cells |
GSE104876 |
Alagille_Nodder |
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Relations |
BioSample |
SAMN07776811 |
SRA |
SRX3275372 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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