|
| Status |
Public on May 04, 2023 |
| Title |
Ribosome profiling in Nrde2 knockout mES cells_ replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
embryonic stem (ES) cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem (ES) cells
genetic background: hybrid 129-C57BL/6
genotype: Nrde2 knockout
|
| Treatment protocol |
Mtrex-KO and Nrde2-KO cells were treated with 100 nM 4-hydroxytamoxifen for 3 and 4 days, respectively. Cells expressing proteins fused to FKBP12F36V degron were treated with 500 nM dTAG-13 for 24 hours. The indicated RNA-seq samples were treated with 0.1 mg/ml cycloheximide (CHX) for 4 hours.
|
| Growth protocol |
Mouse embryonic stem cells were grown under standard conditions in medium containing serum, LIF and 2i.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed and digested with Benzonase. Ribosome footprints were isolated as a band migrating slightly slower than a 35 nt ssDNA oligo on a 15 % Novex TBE-Urea polyacrylamide gel. After ribosomal RNA depletion, ribosome footprints were polyadenylated and purified using SPRI beads. For details see Flemr et al, 2021.
RNA was reverse transcribed with SuperScript II (ThermoFisher 18064014) in the presence of a template-switching oligo. The resulting cDNA was purified with SPRI beads and libraries were amplified using NEBNext Ultra II Q5 master mix (NEB M0544L). For a detailed protocol, see Flemr et al, 2021.
BCLIP-seq
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Riboseq_cpm_per_gene.txt
Nrde2KO_Riboseq_r2
|
| Data processing |
Library strategy: Ribo-seq
Samples were pooled and sequenced on an Illumina HiSeq 2500 instrument.
BCLIP-seq and Ribo-seq samples were demultiplexed using pyBarcodeFilter.py. RNA-seq demultiplexing and fastq conversion were performed with bcl2fastq2 v1.17.
BCLIP-seq preprocessed reads (collapsed and adaptor trimmed) were aligned (splicing-aware) to the mouse genome (mm10) including the transcriptome annotation of GENCODE release M23 using STAR version 2.7.0a (Dobin et al., 2013) with the parameters: --outFilterMultimapNmax 20 --outFilterMismatchNoverLmax 0.05
RNA-seq and Ribo-seq reads were mapped (splicing-aware) to the mouse genome (mm10) including the transcriptome annotation of GENCODE release M23 using STAR version 2.7.3a. with the parameters: --outFilterMultimapNmax 100 --outFilterMismatchNoverLmax 0.05 --outSAMmultNmax 1. Counts per million tables were generated using the featureCounts function from the Rsubread package with the parameters: useMetaFeatures=TRUE,allowMultiOverlap=FALSE, minOverlap=5, countMultiMappingReads=FALSE, fraction=FALSE, minMQS=255, strandSpecific=1 for Ribo and 2 for RNAseq.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files were generated by first using samtools to filter out only uniquely mapping reads, then per base coverage was calculated using bedtools, counts were normalized to 1 mio genome mapping reads and bigwigs were generated using bedGraphToBigWig (USCS tools). Bed files containing peak positions were generated from the .narrowPeak output file from MACS2. HiC allValidPairs files were generated by HiC-Pro 2.9.0. 4C wig files for replicates were generated from the filtered_reads output from peak_C by adding a wiggle file header.
|
| |
|
| Submission date |
Jul 22, 2021 |
| Last update date |
May 04, 2023 |
| Contact name |
Fabio Mohn |
| E-mail(s) |
fabio.mohn@fmi.ch
|
| Organization name |
Friedrich Miescher Institute for Biomedical Research
|
| Lab |
Buehler Lab
|
| Street address |
Maulbeerstrasse 66
|
| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE179744 |
Novel RNA-binding Proteins Bind U1 snRNA to Promote Splicing of Weak 5' Splice Sites |
|
| Relations |
| BioSample |
SAMN20353361 |
| SRA |
SRX11525587 |
