|
| Status |
Public on Jul 20, 2020 |
| Title |
mSTARR_pcDNA_1 |
| Sample type |
SRA |
| |
|
| Source name |
BAC DNA spanning Tbx3 locus
|
| Organism |
Mus musculus |
| Characteristics |
strain: FVB/N
transfection: Murine Tbx3 STARR-seq library transfected in COS7 cells
co-transfection: STARR-seq library co-transfected with pcDNA
|
| Treatment protocol |
Cells were transfected with 150μg library DNA and 75μg of pcDNA (control), SG4 (15μg Smad1, 15μg Smad4, 30μg Alk3 and 15μg Gata4 expression vectors) or Wnt (15μg Tcf4 expression vector, 60μg pcDNA, and 0.4M LiCl) using Polyethylenimine 25 kDa (PEI; Sigma-Aldrich; 408727) in a ratio of 1:3 (DNA:PEI). Medium was refreshed 6 hours after transfection. 48 hours after transfection, total RNA was isolated.
|
| Growth protocol |
15*10^6 COS7 or HeLa cells were cultured in DMEM (ThermoFisher Scientific; 31966-021) supplemented with 10% FBS (ThermoFisher Scientific; 10270-106) and 1% Pen/Strep (ThermoFischer Scientific; 15070-063).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
48 hours after transfection, total RNA was isolated using the Qiagen RNeasy maxi prep kit (Qiagen; 75162). polyA+ RNA was isolated using Dynabeads Oligo-dT25 (ThermoFisher Scientific; 61002) and treated with Ambion turboDNase (ThermoFisher Scientifc; AM2238) for 30min at a maximum concentration of 150ng/μl. RNA was purified using the Qiagen RNeasy MinElute kit (Qiagen; 74204).
First-strand cDNA synthesis and library preparation was performed as described (Arnold et al., Science 2013). Library quality and concentration were assessed by Bioanalyzer (Agilent) and KAPA Library Quantification Kit (KAPA Biosystems; KK4824). Libraries were pooled equimolarly before sequencing on a Miseq (PE150).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
mSTARR_pcDNA_1
|
| Data processing |
Library strategy: STARR-seq
STARR-seq reads were mapped to mm9 build of the mouse genome using BWA (Li et al., Bioinformatics 2009).
For visualization purposes, bam files were converted to bigwig files using bamCoverage (Ramirez et al., Nucleic Acids Research 2016) and visualized using the UCSC genome browser (Kent et al., Genome Research 2002).
We compared bam files of m/hSTARR_SG4/Wnt to m/hSTARR_pcDNA using bamCompare (Ramirez et al., Nucleic Acids Research 2016) with bin sizes of 50 bases to compute the log2 of the number of reads ratio. Bins were filtered to exclude regions with a read count <75.
Flanking bins were merged using mergeBED (overlaps on either strand; maximum distance between features: 0) (Quinlan et al., Bioinformatics 2010). Merged bins with a log2 fold change of >0.585 (fold change >1.5) were included for further analysis.
Genome_build: mm9 (MGSCv37)
Supplementary_files_format_and_content: STARR-seq .bigwig files: Normalized read count of sequencing reads mapped to mm9 reference genome.
Supplementary_files_format_and_content: STARR-seq .bed files: Coordinates of genomic regions with a log2 fold ratio of >1.5 of SG4-/Wnt- over pcDNA sequence read signal.
|
| |
|
| Submission date |
Feb 13, 2020 |
| Last update date |
Jul 20, 2020 |
| Contact name |
Vincent M. Christoffels |
| E-mail(s) |
v.m.christoffels@amsterdamumc.nl
|
| Phone |
0205667821
|
| Organization name |
Amsterdam UMC
|
| Department |
Medical Biology
|
| Lab |
Vincent Christoffels
|
| Street address |
Meibergdreef 15 K2-159
|
| City |
Amsterdam |
| State/province |
N/A (NA) |
| ZIP/Postal code |
1105 AZ |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL16417 |
| Series (1) |
| GSE145257 |
Identification of active gene regulatory elements in the human and murine TBX3 locus |
|
| Relations |
| BioSample |
SAMN14096442 |
| SRA |
SRX7719255 |
