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Status |
Public on Mar 08, 2022 |
Title |
STAPseq_BRD4_psAuxin_rep2 |
Sample type |
SRA |
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Source name |
HCT116 cells
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Organism |
Homo sapiens |
Characteristics |
cell line: HCT116 cell type: CRISPR/Cas9 edited cells genotype: OsTir1+/-;Brd4-AID+/+ treatment: auxin (IAA)
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Treatment protocol |
4x10^7 BRD4-AID tagged cells per replicate were electroporated with a STAP-seq library and treated with either water (mock) or 500µM auxin (IAA) for 6h.
|
Extracted molecule |
polyA RNA |
Extraction protocol |
The TATA-box + CCAAT-box promoter candidate library was cloned from a pool of 2,000 synthesized 300-mer oligonucleotides (Twist Biosciences Inc) into a human STAP-seq screening vector (Addgene #). The candidate sequences spanned 240bp in total, with 205bp upstream and 35bp downstream of the selected TSSs, followed by a 10bp random barcode, and flanked by the Illumina i5 (25bp) and i7 (25bp) adapter sequences upstream and downstream, respectively, serving as constant linkers for amplification and cloning. To comprehensively amplify the oligonucleotide pool (diluted to 1ng/µl) and thereby create the candidate library insert we performed 20 PCR reactions (98°C for 45 seconds (s); followed by 15 cycles of 98°C for 15s, 65°C for 30s, 72°C for 10s) with 1µl diluted oligonucleotide pool as template, using KAPA Hifi Hot Start Ready Mix (KAPA Biosystems; cat. no. KK2602) and primers (fw: TAGAGCATGCACCGGACACTCTTTCCCTACACGACGCTCTTCCGATCT and rev: GGCCGAATTCGTCGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT) that add extra 23bp to each of the adapters, serving as homology arms for directional cloning of the candidate library insert using In-Fusion HD (Clontech; cat. no. 639650). All PCR reactions were pooled and purified with Agencourt AMPureXP DNA beads (ratio beads/PCR 1.4; cat. no. A63881). Directional cloning of the library insert (amplified oligonucleotide pool) into the STAP-seq screening vector was performed using In-Fusion HD (Clontech; cat. no. 639650) recombination.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
NextSeq 550 |
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Description |
PCR amplified cDNA (STAP-seq transcript)
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Data processing |
Library strategy: STAP-seq Basecalls where performed using CASAVA 1.9.1 Read alignment: 10nt long unique molecular barcode identifier (UMI) was removed from the 5' end of the first read and kept track of for later quantification. Paired-end reads were mapped to a reference containing 250bp long barcoded sequences of all 2,000 promoter candidates and to M. musculus (mm9) genome (for spike-in controls) using Bowtie version 1.2.2 allowing 1 mis-match. Only unique mappings with reverse reads mapping to the end of the candidate sequence were kept, ensuring they correspond to reporter transcripts transcribed from that particular cloned candidate. For paired-end reads that mapped to the same positions, we collapsed those that have identical UMIs as well as those for which the UMIs differed by 1 nucleotide to ensure the counting of unique reporter transcripts. Quantification and normalization: Tag counts at each position represent the sum of the 5′ ends of UMI collapsed fragments and report the number of unique mRNA molecules initiated at that position. For each dataset, the tag count at every position in each candidate sequence was quantified. We did the same for the 9 spike-in M. musculus promoters, and the counts were used to calculate normalization factors between the datasets. For each spike-in promoter we first assigned factor 1 to the dataset with the lowest count and then calculated down-scaling factors for all other datasets based on their counts for that promoter. Final normalization factors were calculated as median of factors for individual spike-in promoters and were used to normalize raw tag counts for each sample. Genome_build: hg19 Supplementary_files_format_and_content: Tab-delimited text files include information on the candidate sequences (origin, genotype and sequence) and their counts in the cloned input STAP-seq library. For each dataset a file with raw counts per position in each candidate sequence is provided.
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Submission date |
Sep 15, 2021 |
Last update date |
Mar 08, 2022 |
Contact name |
Vanja Haberle |
E-mail(s) |
vanja.haberle@imp.ac.at
|
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 |
|
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Platform ID |
GPL21697 |
Series (2) |
GSE156741 |
Differential cofactor dependencies define functionally distinct types of human enhancers |
GSE184236 |
Differential cofactor dependencies define functionally distinct types of human enhancers (STAP-seq) |
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Relations |
BioSample |
SAMN21445513 |
SRA |
SRX12200068 |