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| Status |
Public on Jan 01, 2017 |
| Title |
S2_GRO5_r1 |
| Sample type |
SRA |
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| Source name |
S2 Cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: S2 (Schneider 2 cells)
tissue: Embryo 20-24h
sample type: 5' Capped Nascent RNA
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| Growth protocol |
Drosophila Schneider S2 cells were grown at 25°C in Shields & Sang M3 media (Sigma; St. Louis, MO) prepared with yeast extract (Sigma; St. Louis, MO) and Bacto Peptone (BD Biosciences; San Jose, CA) supplemented with 10% heat-inactivated FBS.
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| Extracted molecule |
total RNA |
| Extraction protocol |
5’GRO-seq was performed as described previously (Lam et al., 2013). About 10^7 Drosophila S2 nuclei were used for run-on with BrU-labeled NTPs. Fragmented transcripts were incubated with polynucleotide kinase (PNK, NEB) to remove 3’ phosphates. BrU-labeled nascent transcripts were subsequently immunoprecipitated with anti-BrdU agarose beads (Santa Cruz Biotech). For 5’GRO-seq, immunoprecipitated RNA was dephosphorylated with calf intestinal phosphatase (NEB). Then 5’ capped fragments were de-capped with tobacco acid pyrophosphatase (Epicenter). Illumina TruSeq adapters were ligated to the RNA 3’ and 5’ ends with truncated mutant RNA ligase 2 (K227Q) and RNA ligase 1 (NEB), respectively. Reverse transcription was performed with Superscript III (Invitrogen) followed by PCR amplification for 12 cycles. Final libraries were size selected on PAGE/TBE gels to 175–225 bp. GRO-seq was essentially performed as 5’GRO-seq, but the immunoprecipitated RNA was directly de-capped with tobacco acid pyrophosphatase (Epicenter) and subsequently kinased with PNK (NEB) prior to adaptor ligation. 5’GRO-seq was performed as described previously (Lam et al., 2013). About 10^& Drosophila S2 nuclei were used for run-on with BrU-labeled NTPs. Fragmented transcripts were incubated with polynucleotide kinase (PNK, NEB) to remove 3’ phosphates. BrU-labeled nascent transcripts were subsequently immunoprecipitated with anti-BrdU agarose beads (Santa Cruz Biotech). For 5’GRO-seq, immunoprecipitated RNA was dephosphorylated with calf intestinal phosphatase (NEB). Then 5’ capped fragments were de-capped with tobacco acid pyrophosphatase (Epicenter). Illumina TruSeq adapters were ligated to the RNA 3’ and 5’ ends with truncated mutant RNA ligase 2 (K227Q) and RNA ligase 1 (NEB), respectively. Reverse transcription was performed with Superscript III (Invitrogen) followed by PCR amplification for 12 cycles. Final libraries were size selected on PAGE/TBE gels to 175–225 bp. GRO-seq was essentially performed as 5’GRO-seq, but the immunoprecipitated RNA was directly de-capped with tobacco acid pyrophosphatase (Epicenter) and subsequently kinased with PNK (NEB) prior to adaptor ligation.
Illumina HiSeq 2500 system and associated protocol was used. Run type: SE50; Run mode: High Output
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
5'GRO-Seq (nascent RNA TSS)
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| Data processing |
Basecalls performed using CASAVA version 1.8.2
Reads were aligned to the Drosophila melanogaster genome (dm3) using bowtie2 (v2.1.0), keeping only reads that mapped to a unique genomic location (MAPQ > 10).
HOMER (v4.7) was used to process alignment files to generate normalized bedGraphs and bigwigs for the UCSC Genome Browser and TSS using modified version of ChIP-Seq peak finding. HOMER finds TSS from 5'GRO-Seq by looking for strand-specific clusters of initiation sites within 150 bp that have more than 4x the normalized read counts relative to non-5' enriched signal (from traditional GRO-Seq).
Genome_build: dm3
Supplementary_files_format_and_content: bed format (centered on TSS)
Supplementary_files_format_and_content: FPKM for the non-TSS samples: Columns: (1) RefSeq ID (2) chromosome (3) Start (4) End (5) Strand (6) Annotation (7) Sample3 FPKM (8) Sample4 FPKM
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| Submission date |
May 08, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Christopher Benner |
| E-mail(s) |
cbenner@ucsd.edu
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| Organization name |
University of California, San Diego (UCSD)
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| Department |
Medicine
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| Street address |
9500 Gilman Dr. MC 0640
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| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093-0640 |
| Country |
USA |
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| Platform ID |
GPL17275 |
| Series (1) |
| GSE68677 |
Characterization of the TCT motif reveals a crucial role of the core promoter micro-environment in gene regulation |
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| Relations |
| BioSample |
SAMN03701981 |
| SRA |
SRX1033610 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1678908_S2-GRO5-r1.tss.bed.gz |
83.0 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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