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| Status |
Public on Dec 15, 2022 |
| Title |
LibFR013_Twistmix2_input_dev_rep3 |
| Sample type |
SRA |
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| Source name |
oligo library (in STARR-seq vector)
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| Organism |
Drosophila melanogaster |
| Characteristics |
sample type: PCR amplified Plasmid library
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 |
genomic DNA |
| 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.
Oligo libraries were synthesized by Twist Bioscience including 249 bp enhancer sequence and adaptors for library cloning. Following the instructions of NEBNext® DNA Library Prep Reagent Set for Illumina® (NEB; cat. no. E6000L), Illumina Multiplexing Adapters (Illumina Inc; cat. no. PE-400-1001) were ligated and homology arms for In-Fusion® recombination were added by PCR, followed by recombination into the STARR-seq vector. To construct the dCP STARR-seq vector, the sequence between BglII and FseI from the pGL3-Promoter backbone (Promega; cat. no. E1751) was replaced with the Drosophila Synthetic Core Promoter (DSCP), an ORF (sgGFP, Qbiogene, Inc), a ccdB suicide gene flanked by homology arms (used for cloning the genomic enhancer candidates during library generation), and the pGL3’s SV40 late polyA-signal. The hkCP STARR-seq vector is identical to the dCP STARR-seq vector except we replaced the DSCP with the RpS12 core promoter. The In-Fusion® reactions were transformed (MegaX DH10B; Invitrogen), grown in liquid culture and plasmids were isolated. See Neumayr et al., Curr. Protoc. Mol. Biol. 2019.
STARR-seq
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Data processing |
Oligo library UMI-STARR-seq RNA and DNA input reads (150 bp) were mapped to a reference containing 249 bp long sequences containing both wildtype and mutated fragments from the Drosophila library using Bowtie v.1.2.2. We demultiplexed reads by the i5 and i7 indexes and subsequently by the oligo identity, since this library contained oligos from other experiments. Mapping reads with the correct length, strand and with no mismatches (to identify all sequence variants) were kept. Both DNA and RNA reads were collapsed by UMI (10 bp, allowing one mismatch; UMI sequences are in the sequencing read name) to ensure the counting of unique reporter transcripts.
We excluded oligos with less than 10 reads in any of the input replicates and added one read pseudocount to oligos with zero RNA counts. The enhancer activity of each oligo in each screen was calculated as the log2 fold-change over input, using both replicates, using DESeq2. We used the counts of wildtype negative regions in each library as scaling factors between samples. This normalization only changes the position of the zero and consequently does not affect the calculation of log2 fold-changes or the p-values for the statistical tests used.
Assembly: dm3 (custom oligo library)
Supplementary files format and content: Table of all Drosophila melanogaster enhancer sequences and their mutant sequences (n=34186) included in the oligo library, with genomic coordinates, oligo sequence, experiment, mutated and pasted motifs, read counts for each screen and final developmental enhancer activity (log2).
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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 |
GPL22106 |
| Series (2) |
| GSE211654 |
Enhancers display sequence flexibility constrained by transcription factor motif syntax [Drosophila motif pasting STARR-seq] |
| GSE211659 |
Enhancers display sequence flexibility constrained by transcription factor motif syntax |
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| Relations |
| BioSample |
SAMN30413390 |
| SRA |
SRX17154580 |
| 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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