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| Status |
Public on Aug 12, 2016 |
| Title |
S2_GAF_RNAi_NHS_PRO_seq |
| Sample type |
SRA |
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| Source name |
S2 cell line
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2
RNAi treatment: GAF-RNAi
time point: before heat shock (non-heat shock)
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| Treatment protocol |
Cells were untreated or treated with double-stranded RNA against LacZ (control), GAF, HSF or M1BP. For the Heat Shock treatments, an equal volume of M3+BPYE medium (no serum) at 48°C was added to the cells, and the cultures were incubated at 37°C for the desired time.
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| Growth protocol |
Cells were grown in M3+BPYE media supplemented with 10% FBS
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| Extracted molecule |
total RNA |
| Extraction protocol |
Nuclei isolation for PRO-seq: Nuclei isolation was performed using standard protocols. Briefly, cells were spun down, washed in cold 1x PBS once and then resuspended in 1mL of cold Swelling buffer (10mM Tris-Cl pH 7.5, 10% glycerol, 3mM CaCl2, 2mM MgAc2, 0.5mM DTT). After incubation on ice for 10 minutes, cells were transferred to a dounce homogenizer and 9mL of cold Lysis buffer (10mM Tris-Cl pH 7.5, 300mM sucrose, 10mM NaCl, 3mM CaCl2, 2mM MgAc2, 0.1% TritonX-100, 0.5mM DTT) was added. After douncing, the cells were spun down, washed in 10mL of cold Lysis buffer and resuspended in 1mL of cold buffer D (50mM Tris-Cl pH 8.0, 25% glycerol, 5mM MgAc2, 0.1mM EDTA, 5mM DTT). Next, cells were spun down again, resuspended in buffer D and flash frozen in liquid nitrogen.
PRO-seq library construction: PRO-seq libraries were prepared as described previously (Kwak et al., 2013). Briefly, 2×10^7 nuclei were added to 2x Nuclear Run-On reaction mix (10mM Tris-Cl pH 8.0, 5mM MgCl2, 300mM KCl, 1% Sarkosyl, 1mM DTT, 0.05mM each of biotin-11-A/C/G/UTP (PerkinElmer), 0.4 u/µL RNase inhibitor) and incubated for 3 min at 30°C. Total RNA was extracted and fragmented by base hydrolysis in 0.2N NaOH on ice for 10 min, and neutralized by adding 1x volume of 1M Tris-Cl pH 6.8. Fragmented nascent RNA was purified using streptavidin beads, ligated with reverse 3' RNA adapter (5'p-GAUCGUCGGACUGUAGAACUCUGAAC-/3'InvdT/), and biotin-labeled products were enriched by another round of streptavidin bead binding and extraction. For 5' end repair, the RNA products were successively treated with tobacco acid pyrophosphatase (TAP, Epicentre) and polynucleotide kinase (PNK, New England Biolabs). 5' repaired RNA was ligated to reverse 5' RNA adapter (5'-CCUUGGCACCCGAGAAUUCCA-3') before being further purified by a third round of streptavidin bead binding and extraction. RNA was reverse transcribed using the Illumina RP1 primer. TruSeq-barcoded libraries were generated by PCR amplification and products greater than 120 bp were PAGE purified and pooled for sequencing.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
library strategy: PRO-seq
PRO-seq data processing: Raw sequencing reads were processed using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html). Illumina adapters were removed with the fastx_clipper tool and reads were trimmed to 26-mers using fastx_trimmer. Sequencing reads shorter than 15 nucleotides were discarded. fastx_reverse_complement was then used to generate the reverse complement of the sequencing reads, which correspond to the sense strand of nascent RNA in the nucleus. Reads were aligned uniquely to the Drosophila melanogaster dm3 reference genome using Bowtie (Langmead et al., 2009) with up to two mismatches. Replicates were highly correlated and were pooled for further analyses.
Genome_build: dm3
Supplementary_files_format_and_content: PRO-seq processed files: Normalized bedgraph files with the combined data for both replicates. Each entry in the bedgraph file represents the number of reads at each position with base-pair resolution. PRO-seq data is strand specific, so a ‘plus’ and a ‘minus’ strand file are provided for each sample. See the accompanying publication for a detailed description of the normalization method used.
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| Submission date |
Feb 05, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Fabiana Duarte |
| E-mail(s) |
fabiana_duarte@g.harvard.edu
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| Organization name |
Harvard University
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| Department |
Stem Cell and Regenerative Biology
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| Lab |
Buenrostro Lab
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| Street address |
7 Divinity Avenue
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE77607 |
Transcription factors GAF and HSF act at distinct regulatory steps to modulate stress-induced gene activation |
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| Relations |
| BioSample |
SAMN04457863 |
| SRA |
SRX1562308 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2055128_S2_GAF_RNAi_NHS_PRO_seq_norm_minus.bedgraph.gz |
22.3 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM2055128_S2_GAF_RNAi_NHS_PRO_seq_norm_plus.bedgraph.gz |
20.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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