|
| Status |
Public on Nov 19, 2019 |
| Title |
MPRA__mESC__rep2__tfxn1 |
| Sample type |
SRA |
| |
|
| Source name |
mESC cell line
|
| Organism |
Mus musculus |
| Characteristics |
tissue: mESC cell line
experiment: MPRA
|
| Treatment protocol |
Three different passaged of HUES64 cells were transiently transfected in triplicate (2.5 million cells each with 25 µg plasmid using 60 µL LipofectStem). Three different passages of mESC cells were transiently transfected (10 million cells each with 40 µg plasmid using 100 µL Lipofectamine 2000).
|
| Growth protocol |
HUES64 cells were cultured in feeder-free mTeSR1 media and passaged every 5-7 days. mESC cells were cultured in feeder-free 2i media and passaged every 3-5 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from transiently transfected cells was precipitated by phenol-chloroform extraction according to standard protocols. DNase treatment (Worthington) was followed by cDNA synthesis with SuperScript III First-Strand Synthesis System (Invitrogen).
cDNA was subject to library amplification and libraries were cleaned up with AMPure beads (0.6x, 1.6x, 1.0x).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
GFP RNA
|
| Data processing |
Library strategy: MPRA
For MPRA samples, we used cutadapt to remove adapters from the raw reads and trim bases with a Phred score < 20. We then counted barcodes if they exactly matched an 11-bp barcode in the design index as well as the constant upstream 6 bp. All scripts used for subsequent data analysis of processed files can be found at https://github.com/kmattioli/2019__cis_trans_MPRA
For RNA-seq samples, we used Hisat2 to align reads hg19 or mm10. We used FeatureCounts to assign reads to genes in GENCODE v25 (lifted to hg19) and GENCODE vM13 (mm10).
Supplementary_files_format_and_content: Barcode counts and gene featureCounts. The file index.txt.gz contains the chromosomal information for each barcode. This file is also available at: https://github.com/kmattioli/2019__cis_trans_MPRA/blob/master/data/01__design/02__index/index.txt.gz
|
| |
|
| Submission date |
Nov 18, 2019 |
| Last update date |
Nov 20, 2019 |
| Contact name |
Martha L. Bulyk |
| Organization name |
Brigham and Women's Hospital
|
| Department |
Division of Genetics
|
| Lab |
Bulyk Lab
|
| Street address |
77 Avenue Louis Pasteur, Rm 468
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE140574 |
Analysis of regulatory element evolution between human and mouse reveals a lack of cis-trans compensation |
|
| Relations |
| BioSample |
SAMN13320596 |
| SRA |
SRX7176008 |
