|
| Status |
Public on Jul 11, 2018 |
| Title |
anti-Qk7 iCLIP, C2C12 myoblasts (MiSeq pilot) |
| Sample type |
SRA |
| |
|
| Source name |
myoblast
|
| Organism |
Mus musculus |
| Characteristics |
cell type: myoblast
cell line: C2C12
|
| Treatment protocol |
crosslinking was performed by irradiating cells with UV light with 400 mJ in an Aglient Stratalinker
|
| Growth protocol |
C2C12 cells were maintained in DMEM (high glucose) supplemented with 10% FBS; for myotube differentiation, C2C12 myoblasts were allowed to reach ~90% confluence then media were changed to DMEM (high glucose) supplemented with 2% horse serum and cultured an additional 72h with daily media change
|
| Extracted molecule |
total RNA |
| Extraction protocol |
following immunoprecipitation and radiolabeling, protein:RNA complexes were separated by SDS-PAGE then transferred to nitrocellulose where they were extracted from a region corresponding to protein:RNA then extracted with phenol:chloroform:isoamyl alcohol after proteinase digestion
All libraries prepared according to Konig et al J Vis Exp. 2011 30;50 and Huppertz et al Methods. 2014 Feb; 65(3):274-87.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
UUUCAANUU bp 1 to 9 of read1 is UMI
c2c12_qk7b_ls041
|
| Data processing |
Library strategy: iCLIP-Seq
iCLIP 1: based on random bases of internal barcode positions 1-9 of each read, the readIDs were divided into classes (for later use in duplicate removal). Note: positions 4-7 comprised fixed values for each sample
iCLIP 2: each read was searched for the presence of adaptor sequence AGATCGGA and trimmed from that point on. Reads shorter than 30bp after trimming (including internal barcode positions 1-9) were discarded.
iCLIP 3: internal barcode positions 1-9 were trimmed before mapping
iCLIP 4: reads mapping (by Bowtie2) to mouse repeatMasker elements were discarded
iCLIP 5: reads were mapped to mm9 by Tophat, aided by a gtf file based on UCSC Knowngenes, and with parameters --no-novel-juncs, --no-coverage-search
iCLIP 6: for each class of internal random barcode bases from step (1), duplicate read mappings of the same length and position were discarded
iCLIP 7: peaks were called on the dup-removed bam files from step (6) by Clipper (Yeo et al) using default parameters for mm9 and --premRNA.
Genome_build: mm9
Supplementary_files_format_and_content: SupplementalTable_QK_CLIPper_Data.xlsx is an Excel spreadsheet file with one worksheet contaiing peak calls for each sample analyzed by Clipper.
|
| |
|
| Submission date |
Aug 14, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
W. Samuel Fagg |
| E-mail(s) |
sam.fagg@gmail.com
|
| Organization name |
University of Texas Medical Branch
|
| Department |
Surgery/Biochemistry and Molecular Biology
|
| Street address |
301 University Blvd, Department of Surgery, MRB5.150, L21875
|
| City |
Galveston |
| State/province |
TX |
| ZIP/Postal code |
77551 |
| Country |
USA |
| |
|
| Platform ID |
GPL16417 |
| Series (2) |
| GSE102614 |
Determination of direct binding targets of Quaking RNA binding proteins |
| GSE102615 |
Quaking isoform depletion in C2C12 myoblasts; direct binding targets of Quaking RNA binding proteins |
|
| Relations |
| BioSample |
SAMN07503570 |
| SRA |
SRX3091897 |
