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Sample GSM7135715 Query DataSets for GSM7135715
Status Public on May 17, 2023
Title E14-paused, NT-gRNA, rep4
Sample type SRA
 
Source name E14
Organism Mus musculus
Characteristics cell type: embryonic stem cells
cell line: E14
genotype: WT(dCas_Alkbh5)
treatment: control (NT-gRNA)
Treatment protocol Cells were transfected with 1μg lenti-sgRNA-BSD (coding for guide RNAs) and selected with 8μg/mL blasticidin for 3 days. Cells were then paused with 200 nM INK128 as mTor inhibitor for 5 days.
Growth protocol ESCs expressing dCas_Alkbh5 cells were grown on gelatin in FBS/LIF
Extracted molecule polyA RNA
Extraction protocol RNA was extracted from equal number of ESCs (2x10^5 cells), with RNeasy Micro Kit with on-column DNase I digestion (QIAGEN). RNA was spiked with synthetic RNAs from the External RNAs Control Consortium (ERCC) Spike-in Mix1 (Thermo Fisher), with 2µl of 1:100 ERCC dilution added to 10µl of RNA, equivalent to ~1-2µg RNA.
Library preparation was done using the NEBNext Ultra II Directional Library Prep Kit for Illumina with the mRNA Magnetic Isolation Module from 1µg RNA, per manufacturer’s instructions (NEB).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Description 04_dCas
Data processing Libraries were trimmed of adaptors and quality-checked using Trim Galore! v0.4.0, and then aligned to the mm10 transcriptome with ERCC sequences using TopHat2115 v2.0.13. Gene counts were obtained from the featureCounts function of subread (v1.5.0) with options: -t exon -g gene_id.
Raw counts were imported into R, and analyzed by cell number normalization using ERCCs with edgeR (v3.32.1).
Assembly: mm10
Supplementary files format and content: tab-delimited text file with raw counts of all genes (including ERCCs) for all samples.
Supplementary files format and content: tab-delimited text file with expression levels (log2) for all samples with cell number normalization.
 
Submission date Apr 02, 2023
Last update date May 17, 2023
Contact name Evelyne Anne Collignon
Organization name Lunenfeld-Tanenbaum Research Institute
Lab Ramalho-Santos lab
Street address 60 Murray Street, Box42,
City Toronto
State/province ON
ZIP/Postal code M5T 3L9
Country Canada
 
Platform ID GPL19057
Series (2)
GSE202848 m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency
GSE228733 m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency [dCas_RNAseq]
Relations
BioSample SAMN34041293
SRA SRX19844936

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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