|
| Status |
Public on Oct 30, 2018 |
| Title |
293T_3'SS library_SF3B1-WT_RNA-seq_ALL |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T
|
| Organism |
Homo sapiens |
| Characteristics |
cell line/type: HEK293T
minigene library: 3'SS library
sf3b1 mutation: wild-type
|
| Treatment protocol |
HEK293t cells were transiently co-transfected with mini-gene plasmid library along with either SF3B1-WT or SF3B1-K700E plasmid. Mini-gene plasmid library were transiently transfected in isogenic cell lines of mouse embryonic stem cell generated using crispr-cas9.
|
| Growth protocol |
HEK293T cells were grown in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1X penicillin/streptomycin (Gibco). mESC were grown in DMEM (Gibco 11965) supplemented with 15% FBS (Gemini Bio) and 1x penicillin/streptomycin (Gibco) in tissue culture dishes coated with 0.1% gelatin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA-seq cells were harvested 48 hr of transfection using RNAEasy kit (Quiagen) followed by DNAse digestion (Ambion, Turbo) as per manufacturer's instructions. For protein cells were lysed in 1X RIPA buffer ( 10mM Tris-Cl(pH 8.0), 1mM EDTA, 0.5 mM EGTA, 1% Triton X-100. 0.1% sodium deoxycholate) supplemented wuth protease inhibitor ( Roche).
Illumina compatible RNA and DNA libraries were generated using custom primers specific to mini-gene library using 1X NEB high fidelity master mix.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
293T_SF3B1_K700E_RNA_ALL
processed data file:
Minigenes_List.txt.gz
|
| Data processing |
For 293T files, A reference minigene file were made using the R1 and barcoded R2 reads from the DNA seq
The fastq files (Wildtype,Mutant) generated from RNA sequencing were locally aligned using blastn 2.6.0+ with the reference sequence file to get the splice junctions in each minigenes
A single file was created combining the witltype and mutatnt alignemnt results, the splice positions and Splicing fraction was determined fro SA1,SA2,SA_cryptic
Kmers were generated (n=6) of all possible combination and the odds ratio of these Kmers were predicted using custom made scripts
The branch point prediction was done by RNAcofold(ViennaRNA-2.3.5 suit)/LaBrachoR/RNAhybrid-2.1.2
mESC_RNA samples were aligned to mm9 using STAR Aligner
Aligned bam files were used for differential splicing analysis using rMATS
Supplementary_files_format_and_content: tab seperated text files were generated for Human and Mouse
|
| |
|
| Submission date |
Jun 10, 2018 |
| Last update date |
Oct 30, 2018 |
| Contact name |
MANOJ PILLAI |
| E-mail(s) |
MANOJ.PILLAI@YALE.EDU
|
| Organization name |
YALE UNIVERSITY
|
| Lab |
PILLAI LAB
|
| Street address |
300 GEORGE STREET #786
|
| City |
NEW HAVEN |
| State/province |
CT |
| ZIP/Postal code |
06511 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE115547 |
Determination of branch point (BP) usage by wild-type and mutant SF3B1 using a minigene library |
|
| Relations |
| BioSample |
SAMN09389034 |
| SRA |
SRX4190640 |
