|
| Status |
Public on Jan 08, 2019 |
| Title |
POOL1__pMPRA1__input_DNA |
| Sample type |
SRA |
| |
|
| Source name |
plasmid DNA
|
| Organism |
synthetic construct |
| Characteristics |
sample type: plasmid DNA
|
| Treatment protocol |
K562 cells were transiently transfected for 48 hours with a ratio of 1:1 with Xtreme Gene HP (Roche), HeLa and HepG2 both with 3:1 Xtreme Gene HP according to the manufacturer protocol. 4x10e6 K562 cells in 6 cm dish, 1.6x10e6 HeLa or HepG2 cells in 10 cm dish were seeded 12-16 hours prior to the transfection. Cells were counted before the transfection and per 1 x 10e6 cells, 5 µg oligopool were transfected. Media was not changed and transfection efficiency was microscopically determined by GFP expression 48 hours post transfection.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA from transiently transfected K562, HeLa, and HepG2 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).
plasmid sequencing and RNA-seq
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
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/2018__lncRNA_promoter_MPRA
Genome_build: hg19
Supplementary_files_format_and_content: Barcode sequence and count per replicate
|
| |
|
| Submission date |
Jul 24, 2018 |
| Last update date |
Jan 08, 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 |
GPL19604 |
| Series (1) |
| GSE117594 |
High-throughput functional analysis of lncRNA core promoters elucidates rules governing tissue specificity |
|
| Relations |
| BioSample |
SAMN09709469 |
| SRA |
SRX4452455 |
