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| Status |
Public on Dec 15, 2022 |
| Title |
ZnT63Cenhancer libraries rep1 STARR-seq |
| Sample type |
SRA |
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| Source name |
S2 cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2
sample type: PCR amplified cDNA (STARR-seq transcript)
promoter: developmental (DSCP)
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| Growth protocol |
Schneider 2 cells were grown in Schneider’s Medium supplemented with 10% heat inactivated FBS at 27ºC. Cells were passaged every 2-3 days.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
24h after electroporation, total RNA was extracted using the RNeasy Maxi kit (Qiagen; cat. no. 75162), followed by polyA+ RNA isolation using Invitrogen Dynabeads Oligo(dT)25 (scaling up the manufacturer’s protocol accordingly; cat. no. 61005) and DNase treatment with Ambion Turbo DNase (cat. no. AM2239) at a concentration of at most 200 ng/µl for 30 minutes (min) at 37°C. The reactions were then subjected to Qiagen RNeasy MinElute reaction clean-up (cat. no. 74204), for Turbo DNase inactivation and RNA concentration. After reverse transcription and second strand synthesis a unique molecular identifier (UMI) was added to each transcript. This is followed by two nested PCR steps, each with primers that are specific to the reporter transcripts such that STARR-seq does not detect endogenous cellular RNAs. See Neumayr et al., Curr. Protoc. Mol. Biol. 2019.
Random 8nt variant libraries were generated using a PCR approach with degenerate oligonucleotides. Forward primers were designed to anneal directly downstream of the enhancer position of interested followed by 8 degenerate bp (creating 65,536 variants) and another 20 bp complementary stretch. Reverse primers were complementary to the 20 bp 5’ of the degenerate stretch. The STARR-seq vector containing the wildtype enhancer of interest (either ced-6 or ZnT63C) was used as a template for the PCR. The PCR was run across the whole STARR-seq plasmid, followed by DpnI digest and a Gibson reaction that re-circularizes the plasmid. Libraries were grown in 2l LB-Amp (final ampicillin concentration 100µg/mL). Variant libraries of the same enhancer i.e. ced-6 enhancer pos110, pos182, pos230, pos241 and ZnT63C enhancer pos142, pos180, pos210 were pooled to equimolar ratio, together with another synthetic oligo library containing wt enhancer sequences and negative regions.
STARR-seq
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Dedicated bowtie indices were created for each enhancer position’s N8 library and combined with an oligo library of thousands of wildtype enhancers and negative sequences (de Almeida et al., 2022) for normalization, all 249 bp-long sequences. UMI-STARR-seq RNA and DNA input reads (paired-end 150 bp) were mapped to these dedicated bowtie indices using Bowtie v.1.2.2 (Langmead et al., 2009). Since the N8 variants were all positioned in the last 150 nt of each enhancer, we allowed for flexible mapping in the beginning of the fragments to increase the number of mapped reads while keeping high sensitivity for the different enhancer variants. Specifically, we trimmed the forward reads to 36 bp and mapped them to the indices allowing for 3 mismatches; the full 150 bp-long reverse reads were mapped with no mismatches, to identify all sequence variants; paired-end reads with the correct position, length and strand were kept. This mapping strategy was used for both DNA and RNA reads. For paired-end DNA and RNA reads that mapped to the same variant, we collapsed those that have identical UMIs (10 bp, allowing one mismatch) to ensure the counting of unique molecules.
We excluded oligos with less than 5 reads in any of the input replicates and less than 1 read in any of the RNA replicates. The enhancer activity of each sequence in each screen was calculated as the log2 fold-change over input, using all replicates, with DESeq2 (Love et al., 2014). We used the counts of wildtype negative regions in each library as scaling factors between samples.
Assembly: dm3 (custom library)
Supplementary files format and content: Table of all enhancers with random 8nt variants at different positions (n=536279), with genomic coordinates, DNA sequence of variants, read counts and enhancer activity (log2) in each library.
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| Submission date |
Aug 19, 2022 |
| Last update date |
Dec 15, 2022 |
| Contact name |
Bernardo P de Almeida |
| E-mail(s) |
bernardo.almeida@imp.ac.at
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| Organization name |
Research Institute of Molecular Pathology (IMP)
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| Lab |
Stark Lab
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| Street address |
Campus-Vienna-Biocenter 1
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| City |
Wien |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL25244 |
| Series (2) |
| GSE211655 |
Enhancers display sequence flexibility constrained by transcription factor motif syntax [Drosophila random variant STARR-seq] |
| GSE211659 |
Enhancers display sequence flexibility constrained by transcription factor motif syntax |
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| Relations |
| BioSample |
SAMN30413394 |
| SRA |
SRX17155289 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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