|
| Status |
Public on May 10, 2024 |
| Title |
PROseq_Zfp574_6h_AID_depletion_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
mESC
|
| Organism |
Mus musculus |
| Characteristics |
cell line: mESC
genotype: AID-Zfp574; N-terminal in OsTir1 parental cell line (FRT-EF1a-ARF16-HA-P2A-OsTiR1-noXhoI-3xmYC-T2A-mCherry-SV40polyA-F3)
treatment: 6h_depletion_IAA
|
| Treatment protocol |
For each condition, 1x10^7 AID-Zfp574 cells were collected and nuclei were isolated after 6h of 500uM IAA treatment or no treatment (two biological replicates per condition). Spike-in control (S2 Drosophila cells; 1% of mESC cells) were added at the level of nuclei permeabilization step.
|
| Growth protocol |
All mouse experiments presented here were carried out in diploid mouse embryonic stem cells (mESCs) that were derived from originally haploid HMSc2 termed AN3-128. The mESCs were cultivated without feeders in high-glucose-DMEM (Sigma-Aldrich) supplemented with 13.5% fetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 1x Penicillin-Streptomycin (Sigma-Aldrich), 1x MEM non-essential amino acid solution (Gibco), 1mM sodium pyruvate (Sigma-Aldrich), 50 mM β-mercaptoethanol (Merck) and in-house produced recombinant LIF.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
PRO-seq protocol was performed as in Serebreni, L. et al. Functionally distinct promoter classes initiate transcription via different mechanisms reflected in focused versus dispersed initiation patterns. EMBO J 42(10), e113519 (2023) with a single modification: the nuclear run-on was performed at 37 °C for 3 min.
PRO-seq libraries were sequenced in paired-end mode with 36-bp read length. To eliminate PCR duplicates, an 8-bp long unique molecular identifier (UMI) was incorporated at the 5′ end of the reads during the sample processing.
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| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
PROseq_counts_Zfp574_0h_ctl_6h_depletion.txt
PROseq_DESeq2_Zfp574_6h_depletion_vs_0h_ctl.txt
|
| Data processing |
Before mapping, the UMI was separated, and the Illumina adapters were trimmed using cutadapt v.1.18. Only reads with a length greater than 10 bp were then mapped using Bowtie v.1.2.2 26, initially to the mm10 version of the mouse genome. The mapping allowed for up to 2 mismatches and reported only the best alignment (-m 1 --best --strata) for each read. To ensure the counting of unique nascent RNA molecules, reads that mapped to the same genomic location were collapsed based on their UMIs, allowing for up to 1 mismatch. To create the PRO-seq coverage signal with the exact positions of RNA pol II molecules, only the first nucleotide of each read (i.e. the 3’ end of nascent transcripts) was considered and the strand swapped to match the transcription direction. A non-redundant CAGE-corrected gene set was used to count the number of UMI-collapesed 1nt-long mapped PRO-seq reads that overlap them.
Differential analysis was performed using DESeq227 (v.1.22.2).
Assembly: mm10
Supplementary files format and content: PROseq_counts_Zfp574_0h_ctl_6h_depletion.txt contain raw counts for all conditions and replicates.
Supplementary files format and content: PROseq_DESeq2_Zfp574_6h_depletion_vs_0h_ctl.txt contains classical DESeq2 output (see DESeq2 documentation for further details about columns content)
Library strategy: PRO-seq
|
| |
|
| Submission date |
Aug 07, 2023 |
| Last update date |
May 10, 2024 |
| Contact name |
Alexander Stark |
| E-mail(s) |
stark@starklab.org
|
| Organization name |
The Research Institute of Molecular Pathology (IMP)
|
| Lab |
Stark Lab
|
| Street address |
Campus-Vienna-Biocenter 1
|
| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
| |
|
| Platform ID |
GPL21626 |
| Series (2) |
| GSE225972 |
Proteome-scale tagging and functional screening in mammalian cells by ORFtag |
| GSE240285 |
Proteome-scale tagging and functional screening in mammalian cells by ORFtag [PRO-Seq] |
|
| Relations |
| BioSample |
SAMN36877358 |
| SRA |
SRX21285806 |
