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Sample GSM7919144 Query DataSets for GSM7919144
Status Public on Dec 29, 2023
Title mESC, KI, LIF2i, Rep2, Ribo-Seq
Sample type SRA
 
Source name mouse embryonic stem cells
Organism Mus musculus
Characteristics cell type: mouse embryonic stem cells
genotype: eIF2{alpha}A/A (KI)
treatment: LIF/2i
rnasei treatment: no
Treatment protocol mESCs were maintained in serum-free ESC media supplemented with either LIF/2i or LIF for 6 days.
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.
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.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
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
 
Submission date Nov 27, 2023
Last update date Dec 29, 2023
Contact name Mehdi Amiri
E-mail(s) m.amiri.biology@gmail.com
Organization name McGill University
Department Biochemistry
Street address 1160 Pine Avenue West
City Montreal
State/province Quebec
ZIP/Postal code H3A 1A3
Country Canada
 
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
Relations
BioSample SAMN38457810
SRA SRX22659191

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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