 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 23, 2021 |
| Title |
NON_REP1_L002 |
| Sample type |
SRA |
| |
|
| Source name |
Samples EIF3D/L, EIF3F/E and EIF3G/E/F PAR-CLIP, nonactivated Jurkat, replicate 1
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: T cell leukemia
cell line: Jurkat Clone E6-1
molecule: RNA crosslinked to eIF3
|
| Treatment protocol |
50 µM of 4-thiouridine was determined as non-toxic to Jurkat cells over the time course of the PAR-CLIP experiments. Jurkat cells seeded at 8 x 10^5 cells ml-1 were treated with 50 µM of 4-thiouridine for 7 hours. Then, the cells were treated with or without 1X Cell Stimulation Cocktail, containing PMA and IONO (ThermoFisher, Cat. #: 00-4970-93) for 5 hours. Cells were then crosslinked on ice with 365 nm UV irradiation at an energy dose of 0.2 J cm-2.
|
| Growth protocol |
Cells were maintained in RPMI 1640 Medium (ATCC modification) with 10% FBS (VWR Life Science Seradigm) and 0.01% Penicillin-Streptomycin (10,000 U/mL) (ThermoFisher).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cells were pelleted by centrifugation at 100 x g for 15 min at 4 °C, and the pellet was resuspended in three volumes of NP40 lysis buffer (50 mM HEPES-KOH pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% Nonidet P-40 alternative, 0.5 mM dithiothreitol (DTT), 1 Complete Mini EDTA-free Protease Inhibitor Cocktail tablet (Roche)). The cell suspension was then incubated on ice for 10 min, passed through an 18G needle five times, and centrifuged at 13,000 x g for 15 min at 4 °C and RNAs were lightly digested by treatment with MNase (Thermo Scientific) at a final concentration of 0.05 U μl-1 for 20 min at 16 °C. For each PAR-CLIP assay 1000 μL of Dynabeads (Invitrogen) and 800 μL of anti-EIF3B antibody (Bethyl A301-761A) were used. The remaining steps of the PAR-CLIP analysis were performed exactly as described in (Pubmed ID: 25849773, 26463383) with the exception of using MNase at 5 U μL-1 for the on-bead digestion step.
PAR-CLIP cDNA libraries were sequenced on an Illumina HiSeq 2500 platform. To eliminate potential PCR biases during PAR-CLIP library preparation, a random bar code was introduced into the 3’ adapter and all the reads that matched the random barcode were collapsed into single reads.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Samples EIF3D/L, EIF3F/E and EIF3G/E/F PAR-CLIP, nonactivated Jurkat, replicate 1
|
| Data processing |
Library strategy: PAR-CLIP
PAR-CLIP replicate 1 library adaptors have 3' sequence TCGTATGCCGTCTTCTGCTTG. This is preceded by a 5-nt UMI barcode for PCR deduplication. Finally, each eIF3 subunit sample is indicated by a 5-nt barcode.
Barcoding for PAR-CLIP samples: PA=TCACTNNNNN PB=TCATCNNNNN PD=TCCACNNNNN P1=TCCGTNNNNN P2=TCACTNNNNN where NNNNN is the UMI for PCR deduplication
Clusters of overlapping sequence reads mapped against the human genome version hg38 were generated using the PARalyzer software (Pubmed ID: 21851591) incorporated into the PARpipe pipeline (https://ohlerlab.mdc-berlin.de/software/PARpipe_119/, (Pubmed ID: 30517751) with the settings below. Binding sites were categorized using the Gencode GRCh38.p12 GTF annotations (gencode.v21.annotation.gtf), https://www.gencodegenes.org/human/.
Settings used for PARpipe: Conversion = T>C; Minimum read count per group = 5; Minimum read count per cluster = 7; Minimum read count for kde = 3; Minimum cluster size = 11; Minimum conversion locations for cluster = 2; Minimum conversion count for cluster = 2; Minimum read count for cluster inclusion = 1; Minimum read length = 20; Maximum number of non conversion mismatches = 1;
Genome_build: hg38
Supplementary_files_format_and_content: Excel spreadsheets with tabulated data from the PAR-CLIP experiment or RNA-Seq experiment.
|
| |
|
| Submission date |
Dec 20, 2021 |
| Last update date |
Dec 23, 2021 |
| Contact name |
Jamie H D Cate |
| E-mail(s) |
j-h-doudna-cate@berkeley.edu
|
| Phone |
510-541-7235
|
| Organization name |
University of California
|
| Department |
Molecular and Cell Biology/Chemistry
|
| Street address |
2151 Berkeley Way
|
| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720-5230 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE191306 |
Genome-wide mapping of eIF3-RNA interactions in Jurkat cells using PAR-CLIP |
|
| Relations |
| BioSample |
SAMN24251772 |
| SRA |
SRX13458749 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
