|
| Status |
Public on Nov 28, 2021 |
| Title |
Coculture rep2 |
| Sample type |
SRA |
| |
|
| Source name |
MCF7+MEF
|
| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
sample type: Coculture
biological replicate: rep 2
technical replicate: rep1
|
| Treatment protocol |
MEF cells were either cultured alone (single culture) or in co-culture with MCF7 cells (co-culture) for 12 hours. After that, cells were trypisized and sorted by antigen-based magnetic sorting using CD326-magnetic microbeads. CD326 is specifically expressed on the surface of MCF7. CD326-depleted fraction is enriched with MEF cells. mRNAs from MCF7 in MEF cells are detected with RNA-Seq. For detailed protocol, see: 1. Dasgupta S, Gerst JE (2020) A protocol for non-biased identification of RNAs transferred between heterologous mammalian cell types using rna tagging, cell sorting, and sequencing. Methods in Mol.Biol. (Humana Press Inc.), pp 195–214
|
| Growth protocol |
All cell lines were cultured in DMEM (high glucose), supplemented with 10% Fetal Bovine Serum (FBS), 1 mM sodium pyruvate and antibiotics (0.1 mg/mL streptomycin and 10 U/mL penicillin) at 37 °C with 5% CO2
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted using the NucleoSpin RNA Mini kit (Macharey Nagel) and RNA integrity was checked by Agilent Tapestation 2100, while the concentration was measured using a NanoDrop microvolume spectrophotometer and Qubit 2.0 BR assay
RNA-sequencing libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs). 2 µg RNA of each sample were processed in two separate reactions. Libraries were amplified with 8 PCR cycles, and sequenced on a Novaseq 6000 SP1 flowcell with 2x150 bp paired-end reads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Sandipan_MC2
|
| Data processing |
Identification of human reads in the mouse RNA data was done as following: 1. Trimming of adapter and low quality bases with cutadapt v 2.8. 2. Alignment to hg38 with STAR (v 2.7.3a) using --alignEndsType EndToEnd and --outFilterMismatchNoverLmax 0. 3. Uniqly aligned reads with mapped mate were selected, and realigned to the mouse genome (mm10). 4. Reads which were aligned to hg38 with 0 mismatches and with >2 mismatches with mm10 were considered as bonafide human reads. 5. HTseq was applied to count the number of reads par human gene.
Genome_build: mm10, hg38
Supplementary_files_format_and_content: text, gene read count
|
| |
|
| Submission date |
Sep 29, 2021 |
| Last update date |
Nov 28, 2021 |
| Contact name |
Tsviya Olender |
| E-mail(s) |
tsviya.olender@weizmann.ac.il
|
| Organization name |
The Weizmann Institute
|
| Department |
Molecular Genetics
|
| Street address |
234 Herzl St.
|
| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
| |
|
| Platform ID |
GPL25526 |
| Series (1) |
| GSE185002 |
NGS Analysis of human mRNAs in mouse cells following a 12-hr 2D-coculture |
|
| Relations |
| SRA |
SRX12403403 |
