|
| Status |
Public on Oct 12, 2017 |
| Title |
Input Ribo(-) |
| Sample type |
SRA |
| |
|
| Source name |
HeLa transfected with YTHDC1-Flag
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
culture media: DMEM
strain: ATCC CCL-2
|
| Treatment protocol |
Transfections were carried out using Lipofectamine 2000, Lipofectamine LTX (multiple plasmids) or Lipofectamine RNAiMAX according to manufacturers protocol in the absence of antibiotics.
|
| Growth protocol |
HeLa cells (ATCC CCL-2) were cultured in DMEM supplemented with 10% FBS and 1% Pen/Strep. mESCs (CGR8) were cultured in GMEM supplemented with 10% FBS, Non-Essential Amino Acids, 1 mM sodium pyruvate, 1 mM 2-mercaptoethanol and 1,000 U/mL LIF. All cells were cultured at 37 degrees C in 5% CO2 incubators.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
PAR-CLIP was performed as previously described (Xu et al., 2014). Breifly, Flag-tagged YTHDC1 was immunoprecipitated from transfected HeLa cells and treated with Rnase. Bound RNA fragments were isolated with TriZol following Proteinase K digestion and subject to ibrary construction.
RIP was performed according to literature procedure (Peritz et al., 2006) using polyA selected RNA and ribosomal RNA depleted RNA as biological replicates.
HeLa cells were fractionated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to manufacturer protocol with the following modifications: following cytoplasmic isolation, nuclei were washed extensively (4 x 200 μL) with PBS. RNA from each portion was collected using Directzol RNA miniprep (Zymo Research) and treated with DNaseI. Samples were depleted of ribosomal RNA prior to sequencing.
mESC mRNA was isolated using TriZol reagent and purified using poly-T oligo magnetic beads.
PAR-CLIP libraries were constructed using Illumina TruSeq Small RNA (Rep 1) or NEBNext Small RNA (Rep 2) library construction kits. RIP-seq and subcellular RNA-seq libraries were constructed using the Illumina TruSeq Stranded mRNA kit. mESC mRNA libraries were constructed using the NEBNext Ultra Directional RNA library construction kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
RIP input
|
| Data processing |
After adaptor trimming with Cutadapt (v1.4.1) and quality control, 50 bp sequence reads were mapped to the human genome (hg19) using TopHat (v.2.0.11) and Bowtie (v1.0.1). 150 bp reads from paired-end sequencing were mapped directly to the mouse genome (mm9) using TopHat.
PAR-CLIP peaks were called using PARalyzer (v.1.5) with default parameters.
RIP-seq and RNA-seq differential expression was quantified between replicate experiments using Cufflinks (v.2.2.1)
Differential splicing was analyzed using MATS (v3.0.8) (rMATS) below a FDR of 0.05.
Genome_build: hg19 (human), mm9 (mouse)
|
| |
|
| Submission date |
Oct 27, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ian Roundtree |
| E-mail(s) |
iroundtree@uchicago.edu
|
| Organization name |
University of Chicago
|
| Department |
Department of Chemistry
|
| Lab |
Chuan He Lab
|
| Street address |
929 E 57th Street E307
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE74397 |
N6-methyladenosine-Mediated Nuclear Export of Messenger RNA |
|
| Relations |
| BioSample |
SAMN04217915 |
| SRA |
SRX1388016 |
