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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 29, 2023 |
| Title |
mESC, WT, LIF2i, Rep2, Ribo-Seq |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic stem cells
genotype: eIF2{alpha}+/+ (WT)
treatment: LIF/2i
rnasei treatment: no
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| Treatment protocol |
mESCs were maintained in serum-free ESC media supplemented with either LIF/2i or LIF for 6 days.
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| Growth protocol |
mESCs were maintained in a serum-free ESC media (EmbryoMax DMEM (Millipore, SLM-220-B) supplemented with 10% KnockOut™ Serum Replacement (Invitrogen, Cat#10828-028), 1X MEM non-essential Amino Acid Solution (Invitrogen, Cat#11140-050), 1 mM Sodium Pyruvate (Invitrogen, Cat#11360-070), 2 mM GlutaMax (Invitrogen, Cat#35050-061), 100 U/mL Penicillin, 100 mg/mL Streptomycin (Invitrogen, Cat#15140-122), 0.11 mM b-mercaptoethanol (Invitrogen, Cat#21985-023), 1 mM PD0325901 (BioVision, Cat#1643-2) and 3 mM CHIR99021 (Stemgent, Cat#04-0004), and 1000 U/mL ESGRO Leukemia Inhibitory Factor (LIF) (EMD Millipore, Cat# ESG1107) at 37°C with 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were washed twice with ice-cooled PBS containing 100 μg/mL CHX, and lysed. Lysates were incubated on ice for 10 min, followed by centrifugation at 16000 x g for 10 min at 4°C. A portion of supernatants was saved for RNA-Seq. Ribo-Seq samples were digested with MNase or RNAse I (Epicenter) and digested polysomes were loaded on a 10-50% sucrose density gradient and centrifuged at 36,000 rpm for 3 hours at 4°C. Fractions corresponding to monosome peak fractions were collected, pooled, and RNAs were extracted using Trizol.
For Ribo-Seq library prep, we followed McGlincy and Ingolia 2017 ribosome profiling technique with some modification. RNA was electrochemically separated on a 15% TBU-Urea gel, and fragments of 25-40 nt were selected. After gel extraction, rRNA depletion, and dephosphorylation, ligation was performed with pre-adenylated linkers containing Unique Molecular Identifiers (UMIs) and barcodes. The ligated samples underwent gel purification, reverse transcription, and circularization using CircLigase I. Another rRNA depletion step was done before qPCR optimization. The final PCR product, confirmed for size and quantity, was sequenced on an Illumina NovaSeq 6000 System.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Sequenced libraries were trimmed using Cutadapt 3.4 by removing the constant adapter (AGATCGGAAGAGCAC) from reads without allowing any mismatches. Any reads shorter than six nt and without the adapter were discarded.
Trimmed reads were demultiplexed using the sample barcode anchored to the 5’ end of reads without allowing any mismatch in the barcodes and deposited here.
The multiplexed reads were then processed for alignments to the mouse transcriptome using the following criteria: First, low-quality reads and reads shorter than 25 were discarded, then reads were sequentially aligned to cytoplasmic and mitochondrion rRNA and tRNA using Bowtie 1.3.0, allowing three mismatches in the reads (parameters: -v 3 --norc -p 12).
The remaining reads were aligned to mouse Gencode version M25 comprehensive transcriptome with Bowtie 1.3.0 allowing two mismatches with the following parameters:(--norc -a -p 12 -m 100 -n 2 --seedlen 25). SAM files were converted to BAM files, sorted, indexed, and then PCR duplicates were removed using UMI-Tools 1.0.1.
The final BAM file was converted to an SQLite file compatible with Trips-Viz (https://trips.ucc.ie/), a platform for the Ribo-Seq data analysis and read count table function was used to extract the read counts for each sample.
Assembly: Gencode version M25 comprehensive transcriptome
Supplementary files format and content: comma separated file includes the raw read_count for each sample
Library strategy: Ribo-Seq
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| Submission date |
Nov 27, 2023 |
| Last update date |
Dec 29, 2023 |
| Contact name |
Mehdi Amiri |
| E-mail(s) |
m.amiri.biology@gmail.com
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| Organization name |
McGill University
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| Department |
Biochemistry
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| Street address |
1160 Pine Avenue West
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| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3A 1A3 |
| Country |
Canada |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE248724 |
Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells [Ribo-Seq] |
| GSE248729 |
Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells |
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| Relations |
| BioSample |
SAMN38457820 |
| SRA |
SRX22659180 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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