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Sample GSM2050802

Query DataSets for GSM2050802

Status

Public on Jun 23, 2017

Title

S2_tandem_HS_R4

Sample type

SRA

Source name

S2, heat shock

Organism

Drosophila melanogaster

Characteristics

cell line: S2 (Schneider cells)
treatment: heat shock
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