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Sample GSM2107431 Query DataSets for GSM2107431
Status Public on Apr 30, 2019
Title Sample_P493tet_1_RF
Sample type SRA
 
Source name P493 cell line
Organism Homo sapiens
Characteristics cell line: P493
molecule type: Ribosome protected 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
 
Description Ribosome protected RNA
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 SAMN04604720
SRA SRX1676732

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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