|
| Status |
Public on Apr 30, 2019 |
| Title |
Sample_P493tet_2_RNA |
| Sample type |
SRA |
| |
|
| Source name |
P493 cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: P493
molecule type: Total RNA
treatment: tetracycline (0.1 uM)
|
| Treatment protocol |
Human B-cell lymphoma P493 cells were treated with Tetracycline (0.1 uM) for 24 hrs followed by cycloheximide treatment for 10 minutes.
|
| Growth protocol |
Human P493 cells were grown in RPMI-1640 supplemented with 10% FBS and 1% Pen/Strep Cells were kept at 37 ¡C in humidified 5% CO2 in air.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA and ribosome protected fragments were isolated following published protocol ( Ingolia, et., al., Nature Protocols 2012).
RNA libraries were prepared for sequencing using standard Illumina protocols. Ribosome protected fragments were prepared for sequencing using Ribosome footprinting protocole described in Ingolia, et., al., Nature Protocols 2012.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Deep sequencing libraries were generated from these fragments and sequenced on the HiSeq 2000 platform. Genome annotation was from the Ensembl database release 75 (http:// www.ensembl.org/).
Ribosome footprint (RF) reads were aligned to reference genome GRCh37 using STARÊ. STAR clips the linker sequence (5Õ- CTGTAGGCACCATCAAT-3Õ), which is technically introduced during RF library construction, and trims the remaining sequence from the 3Õ end while aligning the reads to reference sequence. Briefly, we set the parameters for PALMapper as follows: maximum number of mismatches: 2; minimum aligning length: 15; maximum intron length (splice alignment): 10000. We only use the uniquely aligned reads for further analysis.
To remove ribosome RNA, the footprint reads were also aligned to a ribosome sequence database using STAR with the same parameters except allowing splice alignment. We retrieved the human ribosome sequences from SILVA35 databases. The FASTA file was used as reference sequence to align against. The rRNA-aligned reads were filtered out from GRCh37-aligned reads.
After removing the rRNA, we still observed a portion of reads that were dominated by linker sequence and Illumina P7 adapter. These reads can also be trimmed during mapping and cause false alignment. Therefore, we searched a string of 1~8 nt from linker sequence around the trimming site (±2 bp) allowing 1 nt mismatch. We removed the read if there was no such linker sequence. Finally, we filtered out reads ² 24-bp and ³ 36-bp, and the remaining reads with aligned length from 25- to 35-bp were used to analyse the translational effects of Myc.
Total mRNA sequencing reads were aligned to the GRCh37 reference using STAR. We performed the splice alignment and only use the uniquely aligned reads with maximum 3 mismatches. rRNA contaminating reads were also filtered out using the same strategy described before.
Genome_build: GRCh37
Supplementary_files_format_and_content: RiboDiff analysis of Ribosome footprint and total RNA libraries generated from human B-lymhoma cell line P493 in Myc On and Off samples is provided as a txt file.
|
| |
|
| Submission date |
Apr 03, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Kamini Singh |
| E-mail(s) |
kamini.singh@einsteinmed.edu
|
| Phone |
7184302466
|
| Organization name |
Albert Einstein College of Medicine
|
| Department |
Molecular Pharmacology
|
| Lab |
Kamini Singh
|
| Street address |
1300 Morris Park Ave, Golding 203
|
| City |
Bronx |
| State/province |
NY |
| ZIP/Postal code |
10461 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE79864 |
MYC dependent mRNA translation shapes gene expression and cell biology |
|
| Relations |
| BioSample |
SAMN04604716 |
| SRA |
SRX1676728 |
