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Sample GSM4458896 Query DataSets for GSM4458896
Status Public on Feb 04, 2022
Title HEK293T rna-seq from guide1_2
Sample type SRA
Source name HEK293 Flp-In T-REx embryonic kidney cell line
Organism Homo sapiens
Characteristics cell line: HEK293T
genotype/variation: HDLBP knockout guide 1
replicate: replicate 2
molecule subtype: poly(A)+ RNA
Treatment protocol Cells were washed with ice cold PBS containing 100 µg/ml cycloheximide. The PBS was thoroughly removed and plates were put on liquid nitrogen for 10 sec and subsequently on ice.
Growth protocol HEK293 and HEK293 HDLBP knockout cell lines (1 10 cm dish per replicate) were grown in DMEM + 10%FBS to ~90 % confluency.
Extracted molecule polyA RNA
Extraction protocol Cells were lysed in mammalian polysome buffer and RNA was extracted using Trizol LS
Illumina TruSeq Stranded mRNA Kit
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description polyA RNA from input lysate
Data processing Basecalls were converted to fastq files using Bcl2Fastq ( Reads were demultiplexed and adapter sequences removed by Flexbar (2.5). Reads were then collapsed to remove PCR duplicates, followed by removal of random nucleotides (four on both 5’ and 3’ end of the reads) using fastx_trimmer (FASTX Toolkit 0.0.14). Reads aligning to rRNA sequences and other sources of contamination using a custom index were removed by Bowtie2 (v2.3.2) and the remaining sequences were aligned to the human transcriptome (hg19) using STAR aligner (2.5.3a) using GTF annotation file Gencode v19 by retaining only the reads that uniquely mapped to the hg19 genome (parameters --outFilterMultimapNmax 1 --outFilterMismatchNmax 5 --outFilterMatchNmin 15 --alignSJoverhangMin 5 --seedSearchStartLmax 20 --outSJfilterOverhangMin 30 8 8 8 --quantMode TranscriptomeSAM). Transcriptome BAM files were converted to the bed format using BedTools bamToBed (v2.26.0). Bed files were then input into riboWaltz (v1.1.0) (PMID:30102689), which we used for downstream quality control and P-site coverage analysis.
To calculate translation efficiencies per gene we used RSEM (v1.2.2) which supplied us with read counts and TPM values per gene. Differences in translational efficacy, as well as in mRNA abundance and due to both effects (transcription and translation) were detected by DESeq2 (1.18.1) with an interaction term model as described previously (PMID: 31763789). Briefly, RPF read counts were normalized using the DESeq2 estimateSizeFactors function by taking into account all read counts. DESeq2 was run with default parameters. Log2-transformed fold changes for downstream comparisons were taken directly from the DESeq2 output.
3’ adaptor 4N-RA3, rApp-NNNNTGGAATTCTCGGGTGCCAAGG-InvdT; 5’ adaptor OR5-4N, rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrCrNrNrNrN
Genome_build: hg19
Supplementary_files_format_and_content: processed_data_riboseq_te.txt: tab delimited matrix of transcript per million (TPM) values per gene for ribosome profiling and rna-seq samples, translational efficiency (te) for all samples and log2-transformed fold changes and DESeq2 p-adjusted values between samples (ribo.rna=change in translational efficiency, rpf=change in ribosome protected fragments).
Supplementary_files_format_and_content: processed_data_riboseq_psite_coverage.txt: tab delimited matrix of ribosome raw and normalized (norm. prefix) P-site coverage per psite position within transcript, psite transcript coordinate (psite), start and stop codon transcript coordinate and distance of psite from start and stop codons in nucleotides.
Submission date Apr 07, 2020
Last update date Feb 04, 2022
Contact name Markus Landthaler
Organization name MDC Berlin
Department Berlin Institute of Medical Systems Biology
Street address Robert-Roessle Str. 10
City Berlin
ZIP/Postal code 13125
Country Germany
Platform ID GPL18573
Series (2)
GSE148258 Vigilin/HDLBP promotes translation of endoplasmic reticulum-targeted mRNAs (WT vs HDLBP Ribo-seq + RNA-seq)
GSE148262 HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins
BioSample SAMN14554486
SRA SRX8077282

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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