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Sample GSM2050769 Query DataSets for GSM2050769
Status Public on Jun 23, 2017
Title S2_tandem_Fl_30_R1
Sample type SRA
 
Source name S2, flavopiridol inhibition
Organism Drosophila melanogaster
Characteristics cell line: S2 (Schneider cells)
treatment: flavopiridol inhibition
footprint conditions: McviPI and SssI footprint
sample type: amplicon bisulfite sequencing
Treatment protocol Transcription inhibition was performed as previously described (Henriques et al. 2013). Triptolide and/or flavopiridol were added at a final concentration of 10uM(BM to the culture media for the indicated time (0, 2.5, 5, 10, 20, 40 minutes). Heat shock was performed in S2 cells as previously described (Wirbelauer et al. 2005). Temperature was quickly raised by mixing equivalent amount of cell containing medium at 25C(BC with pre-warmed medium (BC). The mix was incubated for 1 min at 37C(BC. After heat shock (HS), cells were let to recover for 5 min.Transcription inhibition (Trp, Fl) inhibition was performed 2.5 minutes prior heat-shock.
Growth protocol Schneider S2 cells were grown at 25 degrees in Schneider's Drosophila medium (LifeTech: 21720-001) supplemented with 10% FBS. OSC cells were grown at 25 degrees in Shields and Sang M3 Insect Medium (Sigma, S8398) supplemented with 1% insulin, 1% glutathione, 10% heat-inactivated FBS, and 10% Fly Extract.
Extracted molecule genomic DNA
Extraction protocol Footprinting protocol was adapted from (Bell et al. 2010; Kelly et al. 2012). 2.5 10^6 Drosophila cells (S2 or OSC) were resuspended in ice-cold lysis buffer (10mM Tris, 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA, 0.5% NP40), incubated 10 min on ice, spun down. Nuclei were washed (10mM Tris, 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA) and resuspended in McviPI reaction buffer (NEB). The nuclei were then incubated with 200U of McviPI (NEB-M0227L) at 30°C for 7.5 min (in presence of 0.6mM SAM, and 300mM Sucrose). The reaction was resuplemented with 100U of McviPI and 128 pmol of SAM before a second incubation round of 30°C for 7.5 min. 10mM MgCl2, 128pmol of SAM and 60U of SssI were added for a third incubation round of 30°C for 7.5 min. Reaction was stopped by adding a SDS-containing buffer (20mM Tris, 600mM NaCl, 1%SDS 10mM EDTA), DNA was extracted after overnight Proteinase K (200 µg/ml) digestion at 55°C. For single treatments, only one of the enzymes was used in the same conditions.
Whole-genome bisulfite libraries were prepared using Illumina TruSeq DNA sample preparation kit following manufacturer recommendation. 5g of sheared DNA (Covaris) was used as an input for end-repair and A-tailing. Methylated adaptors were ligated and large DNA fragments (400-600bp) were selected on a low melting agarose gel (BIO-RAD -161-3107). The extracted material was used as an input for bisulfite conversion (Qiagen - 59104). The converted DNA was amplified by 10 cycles of PCR using Pfu Cx HotStart (Agilent - 600410) using Illumina primers. PCR product was purified using AMPureXP beads (Beckman Coulter - A63880) and controlled on Bioanalyser High sensitivity (Agilent 5067-4626). The samples were run on an Illumina HiSeq 2500 generating 150bp paired-end reads. Amplicon bisulfite sequencing was performed as previously described (Baubec et al. 2015). Bisulfite amplicons libraries were prepared using NEBNext ChIP kit and sequenced on a MiSeq instrument generating 250bp paired-end reads.
 
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina MiSeq
 
Data processing Raw sequence files were preprocessed using Trimmomatic (Bolger et al. 2014) to remove Illumina adaptor sequences, remove low-quality reads and trim low-quality bases.
The trimmed reads were then aligned using QuasR (using Bowtie as an aligner) (Gaidatzis et al. 2014; Langmead et al. 2009) against a bisulfite index.
Context-independent cytosine methylation call was performed using QuasR.
Custom R functions were developed to determine context-dependent (CG, GC) average methylation.
Genome_build: Release 5 (dm3)
Supplementary_files_format_and_content: genome_wide_methylation_average_dm3.txt: Tab-delimited text file providing single C methylation genome wide for S2 and OSC replicates.
 
Submission date Jan 28, 2016
Last update date Mar 23, 2020
Contact name Dirk Schuebeler
Organization name Friedrich Miescher Institute for Biomedical Research
Street address Maulbeerstrasse 66
City Basel
ZIP/Postal code 4058
Country Switzerland
 
Platform ID GPL16479
Series (1)
GSE77369 Genome-wide single molecule footprinting reveals high RNA polymerase II turnover at paused promoters
Relations
BioSample SAMN04447764
SRA SRX1552673

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not applicable for this record

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