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| Status |
Public on Jul 19, 2021 |
| Title |
RING1AKO.RING1BAID_NucRNA_IAA_2h_rep3 |
| Sample type |
SRA |
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| Source name |
Mouse embryonic stem cells with Drosophila SG4 spike-in, auxin-treated (2h IAA)
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| Organisms |
Drosophila melanogaster; Mus musculus |
| Characteristics |
cell type: Mouse embryonic stem cells
genotype: E14 Rosa26::Tir1; Ring1A-/-; AID-Ring1B
replicate: 3
treatment: auxin (IAA)
treatment duration: 2 hours
spike-in reference organism: Drosophila melanogaster
spike-in cell line: SG4
molecule subtype: nuclear RNA
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| Treatment protocol |
To induce AID-RING1B degradation, water-dissolved auxin (indole-3-acetic acid (IAA), Sigma) was mixed with cell medium to the final concentration of 500 µM and added to PRC1deg cells at designated times before harvesting by trypsinisation. To induce dTAG-SUZ12 degradation, PRC2deg cells were treated with 100 nM dTAG-13 compound for 2 hours.
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| Growth protocol |
Mouse embryonic stem cells were grown on gelatin-coated plates, in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies) supplemented with 15% fetal bovine serum (FBS, Labtech), 0.5 mM beta-mercaptoethanol (Life Technologies), 2 mM L-glutamine (Life Technologies), 1x penicillin-streptomycin (Life Technologies), 1x non-essential amino acids (Life Technologies) and 10 ng/mL leukemia inhibitory factor. Cells were cultured at 37°C with 5% CO2. For calibration of genomic experiments, we used either Drosophila S2 (SG4) cells, cultured at 25°C in Schneider’s Drosophila Medium (Life Technologies), supplemented with 10% heat-inactivated FBS and 1x penicillin-streptomycin.
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| Extracted molecule |
total RNA |
| Extraction protocol |
2x107 mouse ESCs (untreated or treated with auxin or dTAG-13 for indicated times) were mixed with 8x106 Drosophila SG4 cells in PBS. Nuclei were released in 1 mL HS Lysis buffer (50 mM KCl, 10 mM MgSO4.7H20, 5 mM HEPES, 0.05% NP40, 1 mM PMSF, 3 mM DTT, 1x PIC) for 1 min at room temperature, and recovered by centrifugation at 1000 g for 5 min at 4°C, followed by three washes with ice-cold RSB buffer (10 mM NaCl, 10 mM Tris pH 7.4, 3 mM MgCl2). Pelleted nuclei were resuspended in 1 mL of TRIzol reagent and RNA was extracted according to the manufacturer’s protocol, followed by treatment with the TURBO DNA-free Kit. Quality of RNA was assessed using 2100 Bioanalyzer RNA 6000 Pico kit (Agilent), followed by rRNA depletion using the NEBNext rRNA Depletion kit (NEB).
RNA-seq libraries were prepared using the NEBNext Ultra (for PRC1deg) or Ultra II (for PRC2deg) Directional RNA Library Prep kit (NEB), indexed using NEBNext Multiplex Oligos (NEB), polled and sequenced as 80 bp (PRC1deg) or 40 bp (PRC2deg) paired-end reads on the Illumina NextSeq 500 in biological triplicates.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
NucRNA_rep3_2h_S10
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| Data processing |
Paired-end reads were first aligned using Bowtie 2 (“–very-fast,” “–no-mixed” and “–no-discordant” options) (Langmead and Salzberg, 2012) against the concatenated mm10 and dm6 rDNA genomic sequence (GenBank: BK000964.3 and M21017.1) in order to discard the reads mapping to rDNA. The remaining unmapped reads were then aligned against the concatenated mm10 and dm6 genome using STAR (Dobin et al., 2013). Reads which failed to map using STAR were further aligned against the concatenated mm10 and dm6 genome using Bowtie 2 (“–sensitive-local,” “–no-mixed” and “–no-discordant” options), to improve the overall mapping of intronic sequences of nascent transcripts present in nuclear RNA fraction. Uniquely mapped reads from the two alignment steps were combined and PCR duplicates were removed using Sambamba (Tarasov et al., 2015).
To internally calibrate RNA-seq, we spiked-in a fixed number of Drosophila SG4 to each experimental sample. For data visualisation, mm10 reads were randomly subsampled using factors that reflected the total number of dm6 reads in each sample.
For differential gene expression analysis, we obtained read counts from the original bam files prior to spike-in normalization for a non-redundant mouse gene set using a SAMtools-based custom Perl script. The non-redundant mm10 gene set (n = 20633) was derived from mm10 refGene genes by filtering out very short genes with poor sequence mappability and highly similar transcripts.
To identify significant gene expression changes following auxin or dTAG-13 treatment, we used a custom R script that incorporates spike-in calibration into DESeq2 analysis (Love et al., 2014) and uses “apeglm” method for LFC shrinkage (Zhu et al., 2019). Spike-in calibration was incorporated by using read counts from a set of unique dm6 refGene genes to calculate DESeq2 size factors for normalization of raw mm10 read counts, as previously described in (Fursova et al., 2019; Taruttis et al., 2017). For a change to be considered significant, we applied a threshold of p-adj < 0.05 and fold change > 1.5.
Genome_build: mm10
Supplementary_files_format_and_content: Stranded bigWig files generated with genomeCoverageBed from BEDTools representing genome coverage for merged biological replicates which have been pre-normalised using spike-in downsampling factors
Supplementary_files_format_and_content: DESeq2 results tables with log2 fold changes shrunken by “apeglm” method and statistical parameters of differential expression gene analysis for comparisons between treated and untreated cells.
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| Submission date |
Oct 12, 2020 |
| Last update date |
Jul 19, 2021 |
| Contact name |
Paula Dobrinić |
| Organization name |
University of Oxford
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| Department |
Department of Biochemistry
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| Lab |
Rob Klose lab
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| Street address |
South Parks Road
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| City |
Oxford |
| ZIP/Postal code |
OX1 3QU |
| Country |
United Kingdom |
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| Platform ID |
GPL25537 |
| Series (2) |
| GSE159399 |
PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency [RNA-seq] |
| GSE159400 |
PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency |
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| Relations |
| BioSample |
SAMN16422273 |
| SRA |
SRX9281860 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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