|
| Status |
Public on Jun 06, 2021 |
| Title |
ES FCS_Rep 3_Luciferase CTL |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic stem cells
cell culture: serum-grown
treatment: luciferase control siRNA
rna prep: rRNA depletion
|
| Treatment protocol |
ESCs were treated for 24hrs with siRNA pools to Stag1 (SA1) including, SmartPool (SP), 5' or 3’ end of Stag1 (5p and 3p) and two sets of control siRNAs, scrambled (SCR) and Luciferase (Luc).
|
| Growth protocol |
Mouse embryonic stem cells (ESC) were cultured in serum (FCS) or naïve (2i) conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was first depleted of rRNA (ES FCS) or enriched for PolyA (ES 2i). Two rounds of rRNA depletion were done.
RNA-seq libraries were prepared using NEBNext Ultra directional RNAseq kit with 8-10 cycles of PCR.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Description |
ES_FCS_All_Reps_Analysis.txt
|
| Data processing |
ES FCS RNA-seq data were processed using the RNA-seq Nextflow pipeline (v19.01.0), with the following parameters –aligner hisat2 –genome mm10, with –reverse_stranded specified for paired-end samples.
FeatureCounts output was parsed through edgeR (v3.16.5) and DESeq2 (v1.14.1) to generate normalized expression counts
The normalized counts for RNAseq Rep1 were calculated in edgeR. Very lowly expressed genes were removed (rowSum cpm <2 across SCR and SA1SP replicates), normalization factors were calculated using calcNormFactors and dispersions estimated using estimateDisp. The edgeR volcano plot statistics were calculated using the exactTest and topTags functions
To generate the normalized counts for all ES FCS RNAseq experiments for GSEA, the FeatureCounts output for all ES FCS experiments was combined into a single table and read it into DESeq2. A DESeq2 object was built using the function DESeqDataSetFromMatrix and estimation of size factors and dispersions were calculated using the DEseq function. Normalized counts were calculated using the ‘counts’ function. Very lowly expressed genes (rowSum normalized count <10 across all samples) were removed
The ES2i raw reads were parsed into VAST-TOOLS to calculate splicing statistics, therefore there is no processed data for them as the raw reads were used. VAST-TOOLS output reported in publication
Genome_build: mm10
Supplementary_files_format_and_content: ES_FCS_Rep1_SA1_SP_KD_Analysis.txt contains the EdgeR normalized counts ES FCS Rep1
ES_FCS_All_Reps_Analysis.txt contains the DESeq2 normalized counts for all ES_FCS experiments
|
| |
|
| Submission date |
Oct 25, 2020 |
| Last update date |
Jun 06, 2021 |
| Contact name |
Suzana Hadjur |
| E-mail(s) |
s.hadjur@ucl.ac.uk
|
| Phone |
+44 7798957199
|
| Organization name |
Ucl
|
| Department |
Cancer biology
|
| Lab |
Genome organization
|
| Street address |
72 Huntley street
|
| City |
London |
| ZIP/Postal code |
Wc1e6bt |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL21493 |
| Series (2) |
| GSE160014 |
STAG proteins mediate heterochromatin organization to support translation and cell identity [RNA-Seq] |
| GSE160390 |
PEZIC_STAG1_Pluripotency |
|
| Relations |
| BioSample |
SAMN16539265 |
| SRA |
SRX9353366 |
