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| Status |
Public on Dec 08, 2021 |
| Title |
11/9 mef2:CD2- RNA |
| Sample type |
SRA |
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| Source name |
sorted embryonic cells depleted of fusion-competent myoblasts
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: reporter insertion pool crossed to mef2[IE-D5]:CD2
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| Treatment protocol |
Single-cell suspensions were prepared from dechorionated embryos and stained with Alexa647-anti-rat CD2 (Biorad MCA154A647), then CD2-positive and -negative populations were sorted.
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| Growth protocol |
Transformant male flies containing random picks from a pool of sequence-tagged reporter constructs were crossed to twi:CD2 or mef2[IE-D5]:CD2 virgin females in population cages. Embryos were collected for 2.25 hours from pre-layed cages and aged 11 hours at 18°.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA and DNA were simultaneously purified from sorted cells, using the back extraction method for DNA. RNA samples were treated with TURBO DNase and reverse-transcribed with a custom primer specific to the reporter gene amplicon.
A single round of primer extension with a custom primer specific to the reporter gene amplicon added an 8-base degenerate UMI sequence, along with the standard Illumina read 1 sequencing primer and Illumina P5 cluster generation primer. After SPRI purification, the target amplicon (including UMI) was amplified with an additional custom primer specific to the reporter gene and the Illumina P5 + read 1 sequencing primer.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
UMI-tagged amplicon containing degenerate sequence tag
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| Data processing |
Reads were processed to remove and retain UMI tags using a custom python script, available upon request.
Common 5' and 3' regions of the remaining sequence were removed using cutadapt 1.14.
The remaining sequence was mapped to a lookup table of degenerate sequence tags using bowtie.
The resulting file of tag-UMI pairs was processed to extract an UMI count using a custom python script, available upon request.
In R 3.5.1, DNA or RNA UMI counts per tag were normalized to total DNA or RNA UMI counts within each replicate and a normalized expression measure (RNA UMI count / DNA UMI count) calculated, then all replicates for one experiment were collected to give a table of individual normalized expression measurements.
In R 3.5.1, tables of all expression measurements within an experiment were filtered to remove individual measurements resulting from <65 DNA UMIs, then to remove enhancers measured in fewer than 4 replicates. Summary statistics (mean and s.e.m.) were calculated for enhancers passing these filters.
Genome_build: n/a
Supplementary_files_format_and_content: tagCounts files contain 4 columns: Enhancer, Tag, UMI count, and Read count
Supplementary_files_format_and_content: fullFiltered files contain 11 columns: Tag, Enhancer_RNA, UMI_count_RNA, Read_count_RNA, Enhancer_DNA, UMI_count_DNA, Read_count_DNA, normUMI_DNA, normUMI_RNA, normUMI, and replicate
Supplementary_files_format_and_content: fullSummaryFiltered files contain 6 columns: Enhancers, numTags, avgUMIcount, sdUMIcount, totalUMIcount, and standardError
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| Submission date |
May 05, 2020 |
| Last update date |
Dec 09, 2021 |
| Contact name |
Stephen Gisselbrecht |
| E-mail(s) |
steveg@alum.mit.edu
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| Phone |
857-540-2853
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| Organization name |
Brigham and Women's Hospital
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| Department |
Division of Genetics, Department of Medicine
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| Lab |
Martha L. Bulyk
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| Street address |
77 Avenue Louis Pasteur, Rm 468
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL19132 |
| Series (2) |
| GSE149906 |
Quantitative-enhancer-FACS-seq (QeFS) reveals epistatic interactions among motifs within transcriptional enhancers in developing Drosophila tissue [Drosophila melanogaster] |
| GSE149908 |
Quantitative-enhancer-FACS-seq (QeFS) reveals epistatic interactions among motifs within transcriptional enhancers in developing Drosophila tissue |
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| Relations |
| BioSample |
SAMN14838112 |
| SRA |
SRX8251651 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4517485_CTW40_tagCounts.txt.gz |
4.4 Kb |
(ftp)(http) |
TXT |
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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