|
| Status |
Public on Feb 04, 2022 |
| Title |
HEK293T ribosome profiling from guide2_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 2
replicate: replicate 2
molecule subtype: ribosome protected 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 |
total RNA |
| Extraction protocol |
Cells were lysed in mammalian polysome buffer, digested with RNase I, loaded on a MicroSpin S-400 HR colum (GE Healthcare) and RNA was extracted using Trizol LS as described in Calviello et al. 2016 (doi:10.1038/nmeth.3688)
libraries were constructed as described in Calviello et al. 2016 (doi:10.1038/nmeth.3688)
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Ribosome profiling (Ribo-seq)
|
| Data processing |
Library strategy: Ribo-seq
Basecalls were converted to fastq files using Bcl2Fastq (2.16.0.10). 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.
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| |
|
| Submission date |
Apr 07, 2020 |
| Last update date |
Feb 04, 2022 |
| Contact name |
Markus Landthaler |
| E-mail(s) |
markus.landthaler@mdc-berlin.de
|
| Organization name |
Max Delbrueck Center
|
| Department |
Berlin Institute for Medical Systems Biology
|
| Street address |
Hannoversche Str. 28
|
| City |
Berlin |
| ZIP/Postal code |
10115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16791 |
| 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 |
|
| Relations |
| BioSample |
SAMN14554490 |
| SRA |
SRX8077278 |
