|
| Status |
Public on May 17, 2023 |
| Title |
E14-paused, NT-gRNA, rep2 |
| 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 |
02_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 |
SAMN34041295 |
| SRA |
SRX19844934 |
